Method for detecting influence of food components on safe medication of picroside

Abstract

The invention particularly relates to a method for detecting the influence of food components on safe medication of picroside. The method comprises the following steps: accurately transferring 2mL of a human serum albumin solution with the concentration of 1 * 10 <-6 > mol L <-1 > into a 1cm cuvette, then adding 4mu L of a food ingredient solution with the concentration of 0.5 * 10 <-3 > mol L <-1 > into the cuvette by using a microsyringe, finally adding 16mu L of a picroside solution into the cuvette by using the microsyringe, uniformly mixing, standing for 2 minutes to obtain a to-be-detected solution, and determining by using a fluorescence spectrometry. The influence of the two food components on safe medication of picroside is researched through fluorescence spectrometry. The method disclosed by the invention has the advantages of simplicity in operation, accurate result, persuasive effect, small sample use amount, simplicity and the like, and lays a foundation for researching the influence of food components on safe medication of picroside.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical methods, and particularly relates to a detection method for analyzing the influence of food components on the safe use of picrosides. Background Art

[0002] Picroside I (PI) and picroside II (PII), as characteristic iridoid glycosides in the traditional medicinal plant Picrorhiza kurroa Royle ex Benth. of the family Scrophulariaceae, have significant pharmacological activities such as antibacterial and anti-inflammatory, hepatocyte protection, and anti-ischemia-reperfusion injury. Their anti-apoptotic properties and biological functions of inhibiting tumor cell migration and regulating the expression of cancer-related proteins highlight the development value of PI / PII as a new anti-cancer candidate drug. In pharmacokinetic studies, human serum albumin (HSA), due to its structural stability and characteristic spectral response, is often used as an ideal model protein for studying drug-protein interactions. The evaluation system of drug clinical efficacy needs to comprehensively consider key parameters such as targeted delivery efficiency, metabolic kinetics characteristics, and bioavailability, and these pharmacokinetic properties are closely related to the drug-HSA binding behavior.

[0003] Methylxanthine alkaloids theophylline (TP) and theobromine (TB), as natural components widely distributed in cocoa products, tea beverages, and processed foods, have seen a significant increase in application in the functional food field in recent years. Pharmacological studies have shown that they can produce coronary artery dilation, diuresis, bronchial smooth muscle relaxation, and positive inotropic effects by inhibiting phosphodiesterase activity, and thus have important application value in the treatment of respiratory diseases.

[0004] The binding of drugs to HSA usually exhibits reversible non-covalent interaction characteristics. When co-ingested food components (such as TP / TB) compete with drugs for HSA binding sites, they may change the free plasma drug concentration through displacement effects, thereby affecting the drug efficacy intensity and metabolic clearance rate. This technical solution aims to clarify the influence law of food-drug interactions on the pharmacokinetic behavior of picrosides by analyzing the molecular interaction mechanism between PI / PII and HSA under the coexistence of TP / TB, providing a scientific basis for optimizing the dietary management of cancer patients and having important guiding significance for improving the clinical drug safety of picrosides. Summary of the Invention

[0005] To solve the above-mentioned existing technical problems, the present invention provides a detection method for the influence of food components on the safe use of picrosides. The fluorescence spectroscopy method is adopted to detect the influence of food components on the interaction between picrosides and HSA.

[0006] The technical solution adopted by the present invention is:

[0007] A detection method for the influence of a food component on the safe use of picroside, comprising the following steps:

[0008] 1) Accurately pipette 2 mL of an HSA solution with a concentration of 1×10 -6 mol L -1 into a 1-cm cuvette, then use a microsyringe to add 4 μL of a food component solution with a concentration of 0.5×10 -3 mol L -1 to the cuvette, and finally use a microsyringe to add 16 μL of a picroside solution to the cuvette. After mixing evenly, let it stand for 2 min to obtain the test solution;

[0009] 2) Take the test solution and scan at a speed of 1200 nm min -1 under the conditions of an excitation wavelength of 280 nm, a fluorescence emission slit width of 5 nm, an excitation slit width of 5 nm, and a temperature of 298 K, and take the fluorescence emission spectrum from 200 to 600 nm;

[0010] 3) Take the test solution and scan at a speed of 1200 nm min -1 under the condition of 298 K, and set the differences between the excitation wavelength and the emission wavelength to be Δλ = 15 nm and Δλ = 60 nm respectively for spectral scanning;

[0011] 4) Under the condition of 298 K, accurately pipette 2 mL of an HSA solution with a concentration of 1×10 -6 mol L -1 into a 1-cm cuvette, then use a microsyringe to add 16 μL of a food component solution with a concentration of 0.5×10 -3 mol L -1 to the cuvette, and finally use a microsyringe to add 16 μL of a picroside solution to the cuvette. After mixing evenly, let it stand for 2 min and scan at a speed of 60000 nm min -1 from an excitation wavelength of 200 to 350 nm and an emission wavelength from 200 to 800 nm for spectral scanning;

[0012] 5) According to whether the binding constant K a value of the picroside - HSA system changes after adding the food component, to judge whether the addition of the food component affects the safe use of drugs; if it changes, it has an impact; otherwise, it has no impact.

[0013] Preferably, in the above detection method, the picroside is picroside I or picroside II.

[0014] Preferably, in the above detection method, in steps 1) and 4), the concentration of the picroside solution is 0.5×10-3 mol L -1 。

[0015] Preferably, in the above detection method, the food component is theophylline or theobromine.

[0016] Preferably, in the above detection method, in step 3), the excitation wavelength is set to 200 - 600 nm.

[0017] The beneficial effects of the present invention are as follows: By studying the effects of TP and TB on the interaction between picroside and HSA through fluorescence spectroscopy, the present invention has the advantages of high sensitivity, strong selectivity, small sample consumption, and simple method. Through fluorescence spectroscopy, the effects of food components such as TP and TB on the safe use of picroside can be accurately judged. Description of the Drawings

[0018] Figure 1 A is the fluorescence spectrum of the system with the change of PI concentration in the presence of TP ([HSA] = [TP] = 1×10 -6 mol / L, [PI] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0019] Figure 1 B is the fluorescence spectrum of the system with the change of PI concentration in the presence of TB ([HSA] = [TB] = 1×10 -6 mol / L, [PI] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0020] Figure 1 C is the fluorescence spectrum of the system with the change of PII concentration in the presence of TP ([HSA] = [TP] = 1×10 -6 mol / L, [PII] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0021] Figure 1 D is the fluorescence spectrum of the system with the change of PII concentration in the presence of TB ([HSA] = [TB] = 1×10 -6 mol / L, [PII] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0022] Figure 2The synchronous fluorescence spectra of HSA in the presence of TP as a function of PI concentration at Δλ = 15 nm and Δλ = 60 nm, the differences between the excitation and emission wavelengths ([HSA] = [TP] = 1×10 -6 mol / L, [PI] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0023] Figure 3 The synchronous fluorescence spectra of HSA in the presence of TB as a function of PI concentration at Δλ = 15 nm and Δλ = 60 nm, the differences between the excitation and emission wavelengths ([HSA] = [TB] = 1×10 -6 mol / L, [PI] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0024] Figure 4 The synchronous fluorescence spectra of HSA in the presence of TP as a function of PII concentration at Δλ = 15 nm and Δλ = 60 nm, the differences between the excitation and emission wavelengths ([HSA] = [TP] = 1×10 -6 mol / L, [PII] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0025] Figure 5 The synchronous fluorescence spectra of HSA in the presence of TB as a function of PII concentration at Δλ = 15 nm and Δλ = 60 nm, the differences between the excitation and emission wavelengths ([HSA] = [TB] = 1×10 -6 mol / L, [PII] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L).

[0026] Figure 6 A is the three-dimensional fluorescence spectrum of HSA-PI in the presence of TP ([HSA] = 1×10 -6 mol / L, [TP] = [PI] = 4.0×10 -6 mol / L).

[0027] Figure 6 B is the three-dimensional fluorescence spectrum of HSA-PI in the presence of TB ([HSA] = 1×10 -6 mol / L, [TB] = [PI] = 4.0×10 -6 mol / L).

[0028] Figure 6 C is the three-dimensional fluorescence spectrum of HSA-PII in the presence of TP ([HSA]=1×10 -6 mol / L, [TP]=[PII]=4.0×10 -6 mol / L).

[0029] Figure 6 D is the three-dimensional fluorescence spectrum of HSA-PII in the presence of TB ([HSA]=1×10 -6 mol / L, [TB]=[PII]=4.0×10 -6 mol / L). Detailed implementation manners

[0030] The present invention will be described in detail below in conjunction with embodiments.

[0031] Example 1 Influence of food components on the safe use of picroside

[0032] The method is as follows:

[0033] (I) Solution preparation

[0034] Preparation of Tris-HCl-NaCl (0.05 mol L -1 , pH = 7.40) buffer solution: Tris stock solution: Weigh accurately 12.115 g of Tris, dissolve it with ultrapure water, and make up the volume to 500 mL in a volumetric flask. HCl solution: Accurately measure 4.165 mL of 12 mol L -1 hydrochloric acid solution, and then make up the volume to 500 mL with ultrapure water in a volumetric flask. Accurately measure 125 mL of Tris stock solution, 210 mL of HCl solution, add 1.4613 g of NaCl, and make up the volume to 500 mL with ultrapure water in a volumetric flask.

[0035] Preparation of HSA solution (1×10 -6 mol L -1 ): Weigh accurately 0.033 g of HAS, dissolve it with Tris-HCl-NaCl buffer solution, and make up the volume to 50 mL in a volumetric flask to obtain an HSA solution with a concentration of 1.0×10 -6 mol L -1 , and store it in a refrigerator at 4°C.

[0036] Preparation of PI solution (0.5×10 -3 mol L -1 ): Weigh accurately 0.012 g of PI, dissolve it with Tris-HCl-NaCl buffer solution, and make up the volume to 5 mL in a volumetric flask.

[0037] Preparation of PII solution (0.5×10 -3mol L -1 ) Preparation: Accurately weigh 0.013 g of PII, dissolve it with Tris-HCl-NaCl buffer solution, and make up the volume to 5 mL in a volumetric flask.

[0038] TP solution (0.5×10 -3 mol L -1 ) Preparation: Accurately weigh 0.9 mg of TP, dissolve it with Tris-HCl-NaCl buffer solution, and make up the volume to 10 mL in a volumetric flask.

[0039] TB solution (0.5×10 -3 mol L -1 ) Preparation: Accurately weigh 0.9 mg of TB, dissolve it with Tris-HCl-NaCl buffer solution, and make up the volume to 10 mL in a volumetric flask.

[0040] (II) Detection and Results

[0041] 1) Accurately pipette 2 mL of HSA solution with a concentration of 1×10 -6 mol L -1 into a 1-cm cuvette, then use a microsyringe to add 4 μL of TP solution with a concentration of 0.5×10 -3 mol L -1 (or TB solution with a concentration of 0.5×10 -3 mol L -1 ) to the cuvette. Finally, use a microsyringe to add 2 μL each time and successively add PI solution with a concentration of 0.5×10 - 3 mol L -1 (or PII solution with a concentration of 0.5×10 -3 mol L -1 ) to the cuvette, so that the concentrations of PI and PII are in the range of 0 - 4×10 -6 molL -1 , with an interval concentration of 0.5×10 -6 mol L -1 . After mixing each solution, let it stand for 2 min to obtain the test solution.

[0042] 2) Take the test solution and scan it at a speed of 1200 nm min -1 under the conditions of an excitation wavelength of 280 nm, a fluorescence emission slit width of 5 nm, an excitation slit width of 5 nm, and a temperature of 298 K, and take the fluorescence emission spectrum in the range of 200 - 600 nm. The results are shown in Figure 1 A- Figure 1 D.

[0043] From Figure 1It can be seen that in the presence of a fixed concentration of TP and TB, as the concentrations of PI and PII increase ([HSA] = [TP] = [TB] = 1×10 -6 mol / L, [PI] / [PII] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0×10 -6 mol / L), the fluorescence intensity of the ternary system gradually decreases, indicating that in the presence of TP and TB, there is an interaction between PI, II and HSA, quenching the intrinsic fluorescence of HSA.

[0044] 3) Take the test solution and scan at a speed of 1200 nm min -1 at 298 K. Set the differences between the excitation wavelength and the emission wavelength to be Δλ = 15 nm (excitation wavelength from 200 - 600 nm, emission wavelength 215 nm) and Δλ = 60 nm (excitation wavelength from 200 - 600 nm, emission wavelength 260 nm) respectively for spectral scanning. The results are as Figures 2 - 5 .

[0045] According to the fluorescence spectrum analysis results ( Figure 2 ), in the co - existence condition of the TP ligand, the interaction between PI and HSA shows a significant dual fluorescence quenching effect: the maximum emission wavelength of tyrosine residues undergoes a slight red - shift, and the characteristic peak intensity of tryptophan residues decreases significantly. This phenomenon indicates that TP enhances the regulatory ability of PI on the conformation of HSA through a synergistic effect, resulting in an increase in the polarity of the micro - environment where aromatic amino acid residues are located and a reconstruction of the hydrophobic cavity structure.

[0046] The synchronous fluorescence spectrum results show ( Figure 3 ) that when TB is used as a competitive ligand, the fluorescence quenching efficiency of HSA induced by PI is significantly improved. This confirms that the combination of TB and PI with HSA further changes the micro - environment of amino acid residues.

[0047] In the system with the presence of the TP ligand ( Figure 4 ), the fluorescence quenching effect of PII on the amino acid residues of HSA is enhanced, indicating that the presence of TP exacerbates the process of change in the local micro - environment of HSA.

[0048] From Figure 5 it can be seen that in the presence of TB, it may change the binding between HSA - PII by forming a ternary complex, thereby enhancing the perturbation intensity of the amino acid micro - environment.

[0049] 4) At 298 K, accurately transfer 2 mL of the HSA solution with a concentration of 1×10 -6 mol L -1 to a 1 - cm cuvette, and then use a microsyringe to add 0.5×10 -3 mol L-1 16 μL of the food ingredient solution, and finally 16 μL of picrorhizide solution was added to the cuvette with a microsyringe. After mixing evenly, it was left standing for 2 min, and scanned at a speed of 60000 nm / min. The excitation wavelength ranged from 200 to 350 nm, and the emission wavelength ranged from 200 to 800 nm for spectral scanning. The results are as -1 follows Figure 6 A - Figure 6 D

[0050] Figure 6 A is the three - dimensional fluorescence spectrum and three - dimensional fluorescence contour map of the HSA - TP - PI system. It can be seen from the figure that Peak 1 and Peak 2 with relatively large peak intensities are the fingerprint characteristic peaks of HSA, located at 225 nm / 340 nm (λ ex / λ em ), 275 nm / 340 nm (λ ex / λ em ), respectively. Peak 1 is formed by the n→π* transition of the carboxyl or amide group of the polypeptide backbone, and Peak 2 is formed by the π→π* transition of tryptophan and tyrosine residues, reflecting the fluorescence characteristics of amino acid residues. After adding TP, the intensities of the two characteristic peaks decreased significantly, indicating the formation of a ground - state complex between them. After adding PI, the fluorescence intensity decreased further significantly, indicating that the presence of TP led to a further change in the HSA conformation induced by PI.

[0051] Figure 6 B is the three - dimensional fluorescence spectrum and three - dimensional fluorescence contour map of the HSA - TB - PI system. Peak 1 is located at 225 nm / 340 nm (λ ex / λ em ), and Peak 2 is located at 275 nm / 340 nm (λ ex / λ em ). With the addition of TB, the intensities of the two characteristic peaks decreased significantly, and the addition of PI further changed the conformation of HSA.

[0052] Figure 6 C is the three - dimensional fluorescence spectrum and three - dimensional fluorescence contour map of the HSA - TP - PII system. When HSA exists alone, the peak value of characteristic peak Peak 1 is 4285, and the peak value of Peak 2 is 4931; after adding TP to form a binary complex, the intensity of the characteristic peak decreased; the addition of PII further quenched the endogenous fluorescence of HSA, and the peak values of Peak 1 and Peak 2 decreased to 3372 and 4095, respectively.

[0053] Figure 6D is the three-dimensional fluorescence spectrum and three-dimensional fluorescence contour map of the HSA-TB-PII system. Due to the addition of PII, the fingerprint characteristic peaks Peak 1 and Peak 2 of the HSA-TB binary system at 225 nm / 340 nm (λ ex / λ em ), 275 nm / 340 nm (λ ex / λ em ) decreased significantly from 4653 and 5281 to 4348 and 4929. PII further changed the original conformation of HSA.

[0054] Based on fluorescence quenching experiments and three-dimensional fluorescence spectrum analysis, this method revealed the regulatory effect of TP / TB on the HSA-picroside system. The experimental data showed that although the introduction of TP and TB did not change the type of fluorescence quenching mechanism of the interaction between picroside (PI / II) and HSA, it significantly affected its binding parameters. Quantitative analysis of the binding constant (K a ) indicated that the binding affinity of the binary system (HSA-PI / II) showed a significant downward trend under coexistence conditions, and the attenuation amplitude of the K a value induced by TP was significantly higher than that of the TB system, suggesting that TP had a stronger interference effect on the stability of the drug-protein complex. The changes in the characteristic parameters of the three-dimensional fluorescence spectrum further corroborated the formation of the ternary system, and the presence of food components exacerbated the protein conformational changes induced by PI / PII.

[0055] The research results of this technology showed that methylxanthine components in the diet might significantly affect the pharmacokinetic properties of picroside, and it was recommended to strictly control the intake of TP during clinical medication. This discovery provided an important experimental basis for clarifying the mechanism of traditional Chinese medicine-food interaction and had practical significance for guiding rational clinical medication.

Claims

1. A detection method for the influence of a food component on the safe use of picroside, characterized in that, It includes the following steps: 1) Accurately pipette 2 mL of HSA solution with a concentration of 1×10 -6 mol L -1 into a 1-cm cuvette, then use a microsyringe to add 4 μL of food component solution with a concentration of 0.5×10 -3 mol L -1 to the cuvette. Finally, use a microsyringe to add 16 μL of picroside solution to the cuvette. After mixing evenly, let it stand for 2 min to obtain the test solution; 2) Take the test solution and scan at a speed of 1200 nm min under the conditions of an excitation wavelength of 280 nm, a fluorescence emission slit width of 5 nm, an excitation slit width of 5 nm, and a temperature of 298 K. -1 Take the fluorescence emission spectrum in the range of 200 - 600 nm. 3) Take the test solution and scan at a speed of 1200 nm min under the condition of 298K -1 Set the differences between the excitation wavelength and the emission wavelength to be Δλ = 15 nm and Δλ = 60 nm respectively, and perform spectral scanning; 4) Under the condition of 298 K, accurately transfer 2 mL of HSA solution with a concentration of 1×10 -6 mol L -1 to a 1-cm cuvette, and then use a microsyringe to add 16 μL of food component solution with a concentration of 0.5×10 -3 mol L -1 to the cuvette. Finally, use a microsyringe to add 16 μL of picroside solution to the cuvette. After mixing evenly, let it stand for 2 min, and then scan at a speed of 60000 nm min -1 from an excitation wavelength of 200 to 350 nm and an emission wavelength of 200 to 800 nm for spectral scanning; 5) According to whether the binding constant K of the picroside-HSA system changes after adding food ingredients, it is determined whether the addition of food ingredients affects the safe use of drugs; if it changes, it is affected; otherwise, it is not affected. a If the value changes, it means there is an impact; otherwise, there is no impact.

2. The detection method according to claim 1, wherein The picroside is picroside I or picroside II.

3. The detection method according to claim 1, characterized in that, In step 1) and step 4), the concentration of the picroside solution is 0.5×10 -3 mol L -1 .

4. The detection method according to claim 1, wherein The food ingredient is theophylline or theobromine.

5. The detection method according to claim 1, wherein In step 3), the excitation wavelength is set to 200 - 600 nm.

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