CN108535213B - Method for evaluating potential use of effective components of cassia twig and monitoring production process - Google Patents
Method for evaluating potential use of effective components of cassia twig and monitoring production process Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Abstract
The invention is a cassia twig effective component latent use appraises and monitoring method of production process, the main content of the former includes using the micro-calorimetric swimming technique to determine the interaction of cinnamaldehyde and human serum albumin, appraise its latent use from the angle of ligand affinity, in order to guarantee accuracy and reliability of the method, have adopted a positive reference substance-ibuprofen and two kinds of compounds-paeoniflorin and glycyrrhizic acid with very good binding capacity of human serum albumin through reporting as the contrast; the main content of the latter is that the real-time monitoring of the cassia twig effective component cinnamaldehyde production process by using the near infrared spectroscopy is used for comprehensively evaluating and analyzing five evaluation indexes of the model in order to ensure the accuracy and the robustness of the established model.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a method for evaluating potential applications of effective components of cassia twig and a method for monitoring a production process of the effective components.
Background
The cassia twig is derived from dried twigs of cinnamon belonging to the family Lauraceae and is a common clinical traditional Chinese medicine. The cinnamaldehyde is the main component of the cassia twig volatile oil, and the study of scholars at home and abroad shows that the cinnamaldehyde has wide pharmacological effects of resisting inflammation, relieving fever and pain, resisting tumors and the like, and has certain treatment effect on a plurality of systemic diseases such as nervous system diseases, cardiovascular diseases, diabetes and the like. And the cinnamaldehyde has small molecular weight, simple structure and easy acquisition, thereby having good drug development prospect.
Human serum albumin is the most abundant protein in plasma, and has good binding and transport capacity to endogenous and exogenous substances, so that many drugs are firstly bound in the blood and then transported to target organs or tissues. The binding process of the drug and the plasma protein may be accompanied by the change of the conformation of the small molecular ligand and the large molecular receptor, and the distribution and the kinetic characteristics of the effector organs of the drug and the plasma protein are influenced to a certain extent by the difference of the number of the acting sites, the specific binding position, the type of the interaction force, the thermodynamic parameters and the like. Therefore, the study of the binding ability and ligand affinity of small molecule ligands to human serum albumin is one of the key indicators for evaluating the potential applications. At present, a plurality of methods for measuring the interaction between small molecules and macromolecules exist, such as surface plasmon resonance, fluorescence polarization technology, dynamic light scattering method and isothermal titration quantitative thermal method, but all have certain limitations, the microcalorimetric electrophoresis technology does not need to change the size and the mass of molecules, does not need to fix samples, needs extremely small amount of samples, is particularly suitable for measuring complex biological samples, and has high measuring sensitivity and good accuracy.
Near Infrared Spectroscopy (NIRS) is a rapid, non-destructive, green and environmentally friendly analytical technique. The spectral region has abundant information content, and most substances respond in the spectral region, so that the holographic. The cinnamaldehyde is used as a main component and an effective component in the cassia twig volatile oil, the development of the cinnamaldehyde can not be separated from the real-time monitoring of the production process, and a good solution is provided for a complex system of the traditional Chinese medicine by the near infrared spectroscopy.
Disclosure of Invention
The invention aims to provide a method for evaluating potential application of cassia twig effective components and a method for monitoring a production process of the cassia twig effective components.
The invention comprises an evaluation method of potential use of effective components of cassia twig and a monitoring method of production process thereof, wherein the evaluation method is characterized in that the method firstly uses micro-calorimetric swimming technique to measure the interaction between cinnamaldehyde and human serum albumin, and evaluates the potential use from the perspective of ligand affinity; the latter is characterized in that near-infrared spectroscopy is applied to real-time monitoring of the production process of the cassia twig effective component cinnamaldehyde.
1. The potential use evaluation method of the cassia twig effective component cinnamaldehyde comprises the following steps:
(1) purchase of experimental materials required for the detection experiment: human serum albumin labeled with Cy5 bio-fluorescent dye at a concentration of 1mg/mL (beijing cyanine mayda technologies ltd.), cinnamaldehyde assay standard (shanghai shidan standard technical service ltd); trident 1M Tris.HCl buffer solution (Biotech, Inc., Bainover, Beijing) at pH 7.4; PEG400 surfactant (bio-technologies ltd, benkyo, benomy);
(2) human serum albumin labeled with Cy5 bio-fluorescent dye at a concentration of 1mg/mL (beijing cyanine mayda technologies ltd.), cinnamaldehyde assay standard (shanghai shidan standard technical service ltd); trident 1M Tris. HCl buffer solution (Biotech, Inc., Bainover, Beijing) at pH 7.4; PEG400 surfactant (bio-technologies ltd, benkyo, benomy);
(3) preparing a solution required by a detection experiment:
preparation of buffer solution: 0.5mL of pH7.4Tris.HCl buffer solution with the concentration of 1.0mol/L and 9.5mL of deionized water are diluted to 5.0mmol/mL for later use; preparing a human serum albumin test solution: 2.0 μ L of 1.0mg/mL human serum albumin was buffered with 5.0mmol/mL Tris.HCl buffer; preparing cinnamaldehyde test solution: taking 1.32mg of cinnamaldehyde standard, adding 0.5mL of PEG400, dissolving with a Tris.HCl buffer solution diluted in the step (r) to a constant volume of 2mL, and taking 5.0mmol/mL as a cinnamaldehyde test stock solution with an initial concentration; preparing a micro-calorimetric phoresis instrument test solution: taking 12 PCR small tubes, numbering 1-12, respectively adding the diluted buffer solution in the step I into No. 2-12 small tubes, then taking 20 mu L of cinnamaldehyde tested stock solution with the initial concentration in the step III, placing the cinnamaldehyde tested stock solution in the step 1 PCR tube, taking 10 mu L from the step 1 PCR tube to the step 2 PCR tube, uniformly mixing, and sucking 10 mu L to the step 3 PCR tube; uniformly mixing the solution in the No. 3 tube, and sucking 10 mu L of the solution into the No. 4 tube; diluting to No. 12 tube according to equal ratio; then taking 10 mu L of prepared human serum albumin test solution into a No. 1-12 tube, and uniformly mixing to obtain MST test solution;
(4) setting parameters of the microcalorimetric phoroptor:
using a micro-calorimetric electrophoresis apparatus to perform pretest on the human serum albumin test solution in the step II, and setting the following measurement parameters according to the pretest result:
receptor Concentration (Target Concentration) 30nM,
the LED lamp color (Excitation Power) is red,
the Power of the LED lamp (Excitation Power) is 40 percent,
40% of infrared laser Power (MST Power);
(5) and (3) determining the affinity of cinnamaldehyde and human serum albumin:
setting the parameters of the instrument according to the result of (3), setting the initial concentration (ligand concentration) of the ligand according to the actual concentration of the cinnamaldehyde, setting the measuring temperature, taking 12 capillaries to absorb the cinnamaldehyde and human serum albumin test samples with the numbers of 1-12 in (2), placing the samples in the instrument to incubate for five minutes, starting to measure, and measuring the interaction of the cinnamaldehyde and the human serum albumin at the temperature of 25 ℃, 30 ℃, 35 ℃, 37 ℃ and 40 ℃ respectively;
(6) data processing and result analysis:
and (4) carrying out data processing and analysis on dissociation constant Kd values at five different temperatures calculated by analysis software.
2. The method for monitoring the production process of the cassia twig effective component cinnamaldehyde comprises the following steps:
(1) purchase of experimental materials:
cassia twig medicinal materials (purchased from Beijing herbal medicine industry Co., Ltd.), cinnamaldehyde standard (Chinese food and drug research institute, batch No. 110710-;
(2) sample preparation and detection:
weighing 7kg of cassia twig medicinal material, placing in a 100L jacketed multifunctional extraction tank, adding 10 times of water, extracting for 2 times, 4h for the first time, and 2h for the second time. In the extraction process, the near infrared spectrum is collected on line at regular intervals, and HPLC detection is carried out at the same time;
(3) and (3) online acquisition of near infrared spectrum:
collecting the spectrum once every 5min in the first decoction heating process; collecting the spectrum every 5min during heating and boiling for 2h, and collecting the spectrum every 10min during 2-4h after heating and boiling; the second decocting and heating process, and collecting the spectrum once every 5 min; collecting the spectrum every 5min for 0-1h after heating and boiling, and collecting the spectrum every 10min for 1-2 h; a total of 65 samples were collected from the above procedure; the spectrum is collected by optical fiber transmission, and the background in the instrument is used as reference. Spectrum collection range: 800-2200 nm, resolution: 0.5nm, 32 times of scanning, room temperature of 20 ℃ and relative humidity of 35 percent;
(4) high performance liquid phase method content determination:
preparation of a reference solution: accurately weighing appropriate amount of cinnamaldehyde and coumarin reference substances, accurately weighing, and adding methanol to obtain 2.0994mg/mL and 0.0992mg/mL solutions respectively;
preparing a test solution: because the concentration of the stock solution of the test sample is high, the stock solution needs to be diluted as follows: taking 2.5mL of sample stock solution in a 25mL volumetric flask during the first decoction heating process, adding 50% methanol for dilution to scale, filtering through a 0.45 mu m filter membrane, and taking the filtrate to obtain the product; in the boiling stage, 2.5mL of sample stock solution is put into a 25mL volumetric flask, 50% methanol is added to dilute the sample stock solution to a scale, and the sample stock solution is filtered by a 0.45-micrometer filter membrane to obtain filtrate; adding 5mL of sample stock solution into a 25mL volumetric flask during the second decoction boiling process, adding 50% methanol to dilute to scale, filtering with a 0.45 μm filter membrane, and collecting the filtrate;
③ chromatographic conditions: a chromatographic column: DIKMA Diamonsil C18 column (250X 4.6mm,5 μm); mobile phase: a is acetonitrile, B is 0.1% glacial acetic acid solution; the elution method is gradient elution, which is shown in table 1; the flow rate is 1.0 mL/min; column temperature: 35 ℃; detection wavelength: 285 nm; injecting 10 mu L of sample;
(5) data processing and near-infrared model construction:
aiming at each component, different preprocessing methods are adopted to establish a full-band Partial Least Squares (PLS) model, and an optimal preprocessing method is selected by taking a cross validation verification Root Mean Square (RMSECV) as an evaluation index; screening a modeling waveband by adopting a combined interval partial least square method (SiPLS), establishing a partial least square model, and evaluating parameters of cross validation Root Mean Square (RMSECV), correction Root Mean Square Error (RMSEC), prediction Root Mean Square Error (RMSEP) and corresponding decision coefficients R2; in order to further verify the reliability of the model, the model is evaluated by adopting a relative error method; the data processing is completed on Unscamblebler data analysis software (version 9.6, Norwegian CAMO software Co.) and MATLAB (version 7.0, U.S. Math Works Co.);
dividing a sample set: dividing 65 sample sets by using a Kennard-Stone method (KS), wherein the divided correction set and verification set are 44 and 21 respectively;
preprocessing the spectrum: comparing the influence of spectrum preprocessing methods such as an original spectrum, a first derivative (1D), a second derivative (2D), a Savitzky-Golay smoothing method (SG), a Multivariate Scattering Correction (MSC), a standard canonical transformation (SNV) and the like on the model performance, adopting an internal cross verification method, and selecting a proper preprocessing method by inspecting the influence of the number of latent variable factors on the square sum of Prediction Residuals (PRESS);
selecting a near-infrared modeling waveband: screening an optimal modeling waveband by adopting a combined interval partial least square method (SiPLS); the parameters of the screening process are as follows: the number of intervals is 20, the number of maximum latent variable factors is 10, and the number of combinations is 3;
establishing and correcting a model: and establishing a PLS model by using normaize on the cinnamaldehyde sample correction set, verifying the model prediction performance by using the internal sample set, and evaluating the model according to the following parameters: the corrected Root Mean Square Error (RMSEC) was 0.0793, the cross-validation Root Mean Square (RMSECV) was 0.0993, the predicted Root Mean Square Error (RMSEP) was 0.0373, the correction set decision coefficient R2cal was 0.9389, the validation set decision coefficient R2val was 0.9085, and the prediction set decision coefficient R2pre was 0.9798.
The method can evaluate the potential application of the cassia twig by measuring the binding capacity of the effective component cinnamaldehyde of the cassia twig and the macromolecular human serum albumin, and can monitor the production process of the cassia twig in real time. The binding rate of the drug plasma protein is one of key indexes for evaluating the potential application of the drug plasma protein, the existing measuring technologies have certain limitations, the micro-calorimetric electrophoresis technology can make up the defects of the technologies, the quantity of consumed samples is very small, the detection limit can reach the nM level, the sensitivity is very high, and the method is particularly suitable for measuring biological samples, so that a simple, convenient and sensitive method is provided for researching the measurement of the affinity of a small molecular ligand and a macromolecular receptor, and the potential druggability of the small molecular ligand and the macromolecular receptor can be better evaluated. In addition, for a multi-component complex system of the traditional Chinese medicine, the near infrared spectroscopy provides a rapid, nondestructive and real-time monitoring method for the production process of the cinnamaldehyde.
The method for evaluating potential application of cinnamaldehyde adopts ibuprofen, two compounds, paeoniflorin and glycyrrhizic acid, which are positive reference substances with good binding capacity with human serum albumin, as reference substances, and mainly comprises the comparison of the interaction of four groups of compounds with human serum albumin at five temperatures of 25 ℃, 30 ℃, 35 ℃, 37 ℃ and 40 ℃ in order to ensure the accuracy and reliability of the measured cinnamaldehyde affinity. In addition, the method for detecting the production process of the cinnamaldehyde, provided by the invention, analyzes five evaluation indexes of the model in order to ensure the accuracy and the robustness of the established model.
Drawings
FIG. 1: results of interaction assay of cinnamaldehyde and human serum albumin at different temperatures
FIG. 2: interaction determination result of ibuprofen and human serum albumin at different temperatures
FIG. 3: interaction determination result of paeoniflorin and human serum albumin at different temperatures
FIG. 4: determination result of interaction between glycyrrhizic acid and human serum albumin at different temperatures
FIG. 5: interaction determination results of four small molecular compounds and human serum albumin at 37 DEG C
FIG. 6: online near infrared spectrum of cassia twig extraction process
FIG. 7: cinnamaldehyde prediction residual sum of squares (PRESS) values under different preprocessing methods
FIG. 8: screening result of cinnamaldehyde modeling waveband
FIG. 9: corresponding result of predicted value and measured value of cinnamaldehyde in cassia twig extract sample
Detailed Description
1. The potential use evaluation method of the cassia twig effective component cinnamaldehyde comprises the following steps:
(1) purchase of experimental materials:
1mg/mL of human serum albumin labeled with Cy5 bio-fluorescent dye (purchased from Beijing Hua Jing Mei Da science and technology Co., Ltd.), ibuprofen powder with purity of more than 98% (purchased from Beijing Hua Jing Hua Mei Da science and technology Co., Ltd.), cinnamaldehyde analysis standard, paeoniflorin analysis standard and glycyrrhizic acid analysis standard (purchased from Shanghai Shidande Standard technology service Co., Ltd.); trident 1M Tris.HCl buffer solution (available from Biotech, Inc., Bainovei, Beijing) at pH 7.4; PEG400 surfactant (from Biotech limited of Baiowei Beijing)
(2) Preparing a detection solution:
preparation of buffer solution: 0.5mL of pH7.4Tris.HCl buffer solution with the concentration of 1.0mol/L and 9.5mL of deionized water are diluted to 5.0mmol/mL for later use;
preparing a human serum albumin test solution: 2.0. mu.L of 1.0mg/mL human serum albumin was buffered with 5.0mmol/mL Tris.HCl buffer
Preparing cinnamaldehyde test solution: taking 1.32mg of cinnamaldehyde standard, adding 0.5mL of PEG400, and dissolving with a Tris.HCl buffer solution diluted in the step I to a constant volume of 2mL to obtain a cinnamaldehyde tested stock solution with an initial concentration of 5.00 mmol/L;
preparing a positive control ibuprofen test solution: taking 1.00mg ibuprofen powder, adding 0.5mL PEG400, dissolving with a Tris.HCl buffer solution diluted in the step I to a constant volume of 2mL, and obtaining an ibuprofen tested stock solution with an initial concentration of 2.40 mmol/L;
preparing a paeoniflorin test solution: dissolving 4.00mg of cinnamaldehyde standard substance with Tris.HCl buffer solution diluted in the step I to a constant volume of 2mL to obtain paeoniflorin tested stock solution with an initial concentration of 8.33 mmol/L;
sixthly, preparing glycyrrhizic acid test solution: taking 13.79mg of cinnamaldehyde standard, dissolving with Tris.HCl buffer solution with pH7.4 to a constant volume of 2mL, and taking 8.38mmol/mL as a tested cinnamaldehyde stock solution with initial concentration;
preparing a micro-calorimetric swimming instrument test solution: taking 12 PCR small tubes, numbering 1-12, respectively adding the diluted buffer solution in the step I into No. 2-12 small tubes, then taking 20 mu L of cinnamaldehyde tested stock solution with the initial concentration in the step III, placing the cinnamaldehyde tested stock solution in the step 1 PCR tube, taking 10 mu L from the step 1 PCR tube to the step 2 PCR tube, uniformly mixing, and sucking 10 mu L to the step 3 PCR tube; uniformly mixing the solution in the No. 3 tube, and sucking 10 mu L of the solution into the No. 4 tube; diluting to No. 12 tube according to equal ratio; then taking 10 mu L of the prepared human serum albumin test solution to a No. 1-12 tube, and uniformly mixing to obtain the test sample of the cinnamaldehyde and the human serum albumin. Preparing mixed test solution of ibuprofen, paeoniflorin, glycyrrhizic acid and human serum albumin by the same method;
(3) setting parameters of the microcalorimetric phoroptor:
using a micro-calorimetric electrophoresis apparatus to perform pretest on the human serum albumin test solution in the step II, and setting the following measurement parameters according to the pretest result:
receptor Concentration (Target Concentration) 30nM,
the LED lamp color (Excitation Power) is red,
the Power of the LED lamp (Excitation Power) is 40 percent,
40% of infrared laser Power (MST Power);
(4) and (3) determining the affinity of cinnamaldehyde and human serum albumin:
setting the parameters of the instrument according to the result of (3), setting the initial concentration (ligand concentration) of the ligand according to the actual concentration of the cinnamaldehyde, setting the measuring temperature, taking 12 capillaries to absorb the cinnamaldehyde and human serum albumin test samples with the numbers of 1-12 in (2) and (2), placing the samples in the instrument to incubate for five minutes, starting to measure, and respectively measuring the interaction between the cinnamaldehyde and the human serum albumin at the temperature of 25 ℃, 30 ℃, 35 ℃, 37 ℃ and 40 ℃;
(5) determination of the interaction of ibuprofen and human serum albumin as a positive control:
determining the interaction of ibuprofen and human serum albumin at 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C and 40 deg.C respectively according to the determination method of cinnamaldehyde in (4);
(6) the comparison products, paeoniflorin and glycyrrhizic acid, and the interaction of the human serum albumin are measured:
measuring the interaction of paeoniflorin and glycyrrhizic acid with human serum albumin at 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C and 40 deg.C respectively according to the method for measuring cinnamaldehyde in (4);
(7) data processing and result analysis:
the results of the tests were processed according to the own analytical Software (NT Analysis Software) and are shown in the following table:
table 1: results of interaction assay of cinnamaldehyde and human serum albumin at different temperatures
Table 2: interaction determination result of ibuprofen and human serum albumin at different temperatures
Table 3: determination result of interaction between glycyrrhizic acid and human serum albumin at different temperatures
Comparing dissociation constants and binding constants of a four-medium molecular compound and human serum albumin at five temperatures, finding that cinnamaldehyde and ibuprofen can be combined with the human serum albumin at the five temperatures, glycyrrhizic acid can be well combined with the human serum albumin at 25 ℃ and 30 ℃, the combination effect is not obvious at 35 ℃, the combination capability is not basically realized at 37 ℃ and 40 ℃, and paeoniflorin can not be combined with the human serum albumin at all temperatures; the four ingredients have the binding capacity of ibuprofen, cinnamaldehyde, glycyrrhizic acid and paeoniflorin from strong to weak in sequence;
② with the temperature rise, the binding ability of the four small molecular substances and the human serum albumin is weaker and weaker (ibuprofen and cinnamaldehyde) and even disappears (glycyrrhizic acid);
according to the binding constants of cinnamaldehyde and ibuprofen with human serum albumin at four temperatures (298K, 303K, 308K and 313K), taking the reciprocal of the temperature (1/T) as an abscissa, and adopting SAS to fit the natural logarithm of the binding constant (lnKa), a straight line of the lnKa about 1/T is obtained, and the results are as follows:
a. the linear equation of lnKa of cinnamaldehyde about 1/T is-40.429 +14744x, R2 is 0.9982, the enthalpy change (delta H) of the combination action is-122.58 kJ/mol, the entropy change (delta S) is-336.13 mol/(K J), and the action types are judged to be hydrogen bonds and van der Waals force according to the literature; the results are shown in Table I;
b. the linear equation of lnKa of ibuprofen about 1/T is-6.9663 +4861.3x, R2 is 0.7879, the enthalpy change (delta H) of the combination is-40.417 kJ/mol, the entropy change (delta S) is-57.918 mol/(K) J, and the acting force types are judged to be hydrogen bonds and van der Waals force according to the literature;
c. the linear equation of lnKa of three temperatures (298K, 303K and 308K) of glycyrrhizic acid for 1/T is-21.11 +7932.4x, R2 is 0.8476, the enthalpy change (Δ H) of the binding action is-65.950 kJ/mol, the entropy change (Δ S) is-175.51 mol/(K × J) according to the equation, and the action types are judged to be hydrogen bonds and van der waals force according to the literature.
2. The method for monitoring the production process of the cassia twig effective component cinnamaldehyde comprises the following steps:
(1) purchase of experimental materials:
cassia twig medicinal materials (purchased from Beijing herbal medicine industry Co., Ltd.), cinnamaldehyde standard (Chinese food and drug research institute, batch No. 110710-;
(2) sample preparation and detection:
weighing 7kg of cassia twig medicinal material, placing in a 100L jacketed multifunctional extraction tank, adding 10 times of water, extracting for 2 times, 4h for the first time, and 2h for the second time; in the extraction process, the near infrared spectrum is collected on line at regular intervals, and HPLC detection is carried out at the same time;
(3) and (3) online acquisition of near infrared spectrum:
collecting the spectrum once every 5min in the first decoction heating process; collecting the spectrum every 5min during heating and boiling for 2h, and collecting the spectrum every 10min during 2-4h after heating and boiling; the second decocting and heating process, and collecting the spectrum once every 5 min; collecting the spectrum every 5min for 0-1h after heating and boiling, and collecting the spectrum every 10min for 1-2 h; a total of 65 samples were collected from the above procedure. Optical fibers are adopted, spectra are collected in a transmission mode, and a built-in background of the instrument is used as a reference; spectrum collection range: 800-2200 nm, resolution: 0.5nm, 32 times of scanning, room temperature of 20 ℃ and relative humidity of 35 percent;
(4) high performance liquid phase method content determination:
preparation of a reference solution: accurately weighing appropriate amount of cinnamaldehyde and coumarin reference substances, accurately weighing, and adding methanol to obtain 2.0994mg/mL and 0.0992mg/mL solutions respectively;
preparing a test solution: because the concentration of the stock solution of the test sample is high, the stock solution needs to be diluted as follows: and 2.5mL of sample stock solution is taken in a 25mL volumetric flask during the first decoction heating process, 50% methanol is added to dilute the sample stock solution to a scale, and the sample stock solution is filtered through a 0.45-micrometer filter membrane to obtain filtrate. In the boiling stage, 2.5mL of sample stock solution is put into a 25mL volumetric flask, 50% methanol is added to dilute the sample stock solution to a scale, and the sample stock solution is filtered by a 0.45-micrometer filter membrane to obtain filtrate; adding 5mL of sample stock solution into a 25mL volumetric flask during the second decoction boiling process, adding 50% methanol to dilute to scale, filtering with a 0.45 μm filter membrane, and collecting the filtrate;
③ chromatographic conditions: a chromatographic column: DIKMA Diamonsil C18 column (250X 4.6mm,5 μm); mobile phase: a is acetonitrile, B is 0.1% glacial acetic acid solution; the elution method was gradient elution, see table 4; the flow rate is 1.0 mL/min; column temperature: 35 ℃; detection wavelength: 285 nm; injecting 10 mu L of sample; the conditions are shown in the following table:
table 4: high performance liquid phase measurement elution scheme table
(5) Data processing and near-infrared model construction:
dividing a sample set: dividing 65 sample sets by using a Kennard-Stone method (KS), wherein the divided correction set and verification set are 44 and 21 respectively;
preprocessing the spectrum: comparing the influence of spectrum preprocessing methods such as an original spectrum, a first derivative (1D), a second derivative (2D), a Savitzky-Golay smoothing method (SG), a Multivariate Scattering Correction (MSC), a standard canonical transformation (SNV) and the like on the model performance, adopting an internal cross verification method, and selecting a proper preprocessing method by inspecting the influence of the number of latent variable factors on the square sum of Prediction Residuals (PRESS); the results are shown in fig. 7 and table 5, with cinnamaldehyde, the best results were obtained by the normaize pretreatment method;
table 5: different pretreatment results of cinnamon twig cinnamaldehyde
Selecting a near-infrared modeling waveband: screening an optimal modeling waveband by adopting a combined interval partial least square method (SiPLS); the parameters of the screening process are as follows: the number of intervals is 20, the number of maximum latent variable factors is 10, and the number of combinations is 3; the results are shown in FIG. 8, the optimal bands of cinnamaldehyde are 1290-1360nm and 1570-1710 nm;
establishing and correcting a model: and establishing a PLS model by using normaize on the cinnamaldehyde sample correction set, verifying the model prediction performance by using the internal sample set, and evaluating the model according to the following parameters: corrected Root Mean Square Error (RMSEC) of 0.0793, cross-validation Root Mean Square (RMSECV) of 0.0993, predicted Root Mean Square Error (RMSEP) of 0.0373, and correction set determination coefficient R2 cal0.9389, a verification set decision coefficient R2 val0.9085 is,Prediction set decision coefficient R2 pre0.9798; the correlation graph of the predicted value of the near infrared spectrum of cinnamaldehyde and the reference value of the high performance liquid phase is shown in figure 9, and the graph shows that the samples are more closely dispersed on two sides of a straight line, which shows that the prediction performance is good.
Claims (4)
1. A potential use evaluation method of cinnamaldehyde is characterized in that the interaction between cinnamaldehyde and human serum albumin is measured by using a micro-calorimetric electrophoresis technology, and the potential use of cinnamaldehyde is evaluated from the perspective of ligand affinity, and the method comprises the following steps:
(1) experimental materials:
human serum albumin and cinnamaldehyde analysis standard substance marked by Cy5 biological fluorescent dye; trident 1mtris. hcl buffer solution; a PEG400 surfactant;
(2) preparing a detection solution:
preparation of buffer solution: taking a Tris.HCl buffer solution with the concentration of 1.0mol/L, and diluting the Tris.HCl buffer solution with deionized water for later use;
preparing a human serum albumin test solution: dissolving human serum albumin in Tris.HCl buffer solution;
preparing cinnamaldehyde test solution: taking a cinnamaldehyde standard substance, adding PEG400, and dissolving with Tris.HCl buffer solution in the first step to a constant volume to obtain a cinnamaldehyde tested stock solution with an initial concentration;
preparing a micro-calorimetric phoresis instrument test solution: taking 12 PCR small tubes, numbering 1-12, respectively adding the diluted buffer solution in the step I into No. 2-12 small tubes, then taking 20 mu L of cinnamaldehyde tested stock solution with the initial concentration in the step III, placing the cinnamaldehyde tested stock solution in the step 1 PCR tube, taking 10 mu L from the step 1 PCR tube to the step 2 PCR tube, uniformly mixing, and sucking 10 mu L to the step 3 PCR tube; uniformly mixing the solution in the No. 3 tube, and sucking 10 mu L of the solution into the No. 4 tube; diluting to No. 12 tube according to equal ratio; then taking the prepared human serum albumin test solution to a No. 1-12 tube, and uniformly mixing to obtain a micro-calorimetric swimming instrument test solution;
(3) setting parameters of the microcalorimetric phoroptor:
pre-testing the human serum albumin test solution in the step II by using a micro-calorimetric electrophoresis apparatus, and setting measurement parameters according to a pre-testing result;
(4) and (3) determining the affinity of cinnamaldehyde and human serum albumin:
setting the parameters of the instrument according to the result of (3), setting the initial concentration of the ligand according to the actual concentration of the cinnamaldehyde, setting the measuring temperature, taking 12 capillaries to absorb the cinnamaldehyde and human serum albumin test sample with the number of 1-12 in (2), placing the sample in the instrument, incubating for a certain time, and starting to measure the interaction between the cinnamaldehyde and the human serum albumin;
(5) data processing and result analysis:
and (3) calculating by using built-in Software NT Analysis Software of the microcalorimetric phorometer to obtain dissociation constant Kd values at different temperatures, and performing data processing and Analysis to obtain the binding capacity and thermodynamic parameters of the cinnamaldehyde and the human serum albumin.
2. The method for evaluating the potential use of cinnamaldehyde according to claim 1, wherein: the pH of the Trident 1M Tris.HCl buffer solution in the step (1) is 7.4.
3. The method for evaluating cinnamaldehyde potential use according to claim 1 or 2, wherein: the concentration range of the buffer solution in the step (2) is 2-10 mmol/ml, the concentration range of the human serum albumin test solution in the step (2) is 3-7mmol/ml, the concentration range of the cinnamaldehyde test stock solution with the initial concentration in the step (2) is 2-10 mmol/ml, and the volume range of the human serum albumin test solution in the step (2) is 10-30 mu L.
4. The method for evaluating cinnamaldehyde for potential use according to any one of claims 1 to 2, wherein: the incubation time in the step (4) is more than or equal to 5 minutes, and the measuring temperature range is 15-45 ℃.
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