CN100565186C - The interaction of medicine and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof - Google Patents
The interaction of medicine and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof Download PDFInfo
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- CN100565186C CN100565186C CNB2006100380701A CN200610038070A CN100565186C CN 100565186 C CN100565186 C CN 100565186C CN B2006100380701 A CNB2006100380701 A CN B2006100380701A CN 200610038070 A CN200610038070 A CN 200610038070A CN 100565186 C CN100565186 C CN 100565186C
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Abstract
The interaction of medicine and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof, step are that employing Raman spectrometer or Fourier Raman spectrometer carry out collection of illustrative plates to drug sample to be measured and measure; Ownership is also selected the absorption band or the absorption peak of sample collection of illustrative plates Chinese traditional medicine, select specific vibrational band as feature with reference to bands of a spectrum; Collect or calculate bands of a spectrum wave number, parameters such as spectral bandwidth, band intensity, bands of a spectrum shape coefficient; Application standard contrast or working curve, qualitative, quantitatively discern and analyze medicine in different systems or inclusion state in the preparation and inclusion degree, dissolving out capability, permeance property and the pharmacokinetic parameters of prediction medicine.The present invention is simple and efficient, and specimen is untouchable, non-destructive, detection sensitivity height, time short, the sample aequum is little and sample need not characteristics such as preparation, has high academic value and actual application value.
Description
Technical field
The present invention relates to a kind of materia medica analytical approach of cyclodextrin inclusion compound, the interaction of especially a kind of medicine and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property parameter thereof.
Background technology
Cyclodextrin has outer hydrophilic, interior hydrophobic cage structure, whole molecule of lipophilicity object or lipophilic structure can be by hydrone and the so-called inclusion compound of its formation (inclusion complexes) in the displacement CD cavity, the i.e. supermolecules of object medicine and main body CD formation.On molecular level, be also referred to as " molecular microcapsule ".Medicine/cyclodextrin supermolecule application purpose mainly is: increase the water-soluble of medicine; Improve stability of drug; Promote drug absorption, improve bioavailability of medicament; Alleviate stimulation and the spinoff of medicine to body; Carrier material as controlled release and targeting preparation; Improve the drug loading aspect of liposome, nanosphere, further improve the function of transmission medicine etc.(Nimodipine, brufen are extremely low because of its solubleness in aqueous solution, the aspect such as therefore absorb and all exist difficult point in formulation preparation, body for insoluble drug.After existing bibliographical information adopted inclusions such as HP-, sulphur butyl ether-beta-schardinger dextrin-both at home and abroad, the water-soluble and bioavilability of insoluble drug was significantly increased, and shows that employing can improve the performance of preparation.Medicine/cyclodextrin supermolecule is the key component in the cyclodextrin formulations, and its inclusion mode and inclusion degree direct influence are to the dissolving out capability and the absorption of medicine.Particularly, the assay determination of inclusion degree or inclusion rate (complexation efficiency) and pharmaceutical property parameter (stripping parameter, permeability parameters, pharmacokinetic parameters) is the important step of design medicament prescription, improvement preparation technology, control product quality.Conventional method of analysis at present such as powder X-raydiffractometry (PXRD), differential scanning calorimetry (DSC), infraredspectra (IR), methods such as NHR, the effect that can only judge inclusion compound Chinese traditional medicine and cyclodextrin whether, when particularly lower the or medicine of system Chinese traditional medicine content is present in the multi-component complex system, the poor accuracy as a result that uses above analytical approach to provide, and generally can't accurately discern system inclusion degree change, can't quantitative test inclusion compound inclusion degree, to the sign of combination in the system also limitation very.Particularly in pharmaceutical preparation, be subjected to all multifactor disturbing effects such as low content, auxiliary material, test condition of medicine, use above analytical approach to be difficult to judge the effect situation of medicine and cyclodextrin, more can't quantitative test medicine/cyclodextrin between the inclusion degree.
Along with the widespread use of cyclodextrin in field of pharmacology, press for and a kind ofly can analyze, characterize inclusion relation, inclusion degree between medicine and the cyclodextrin and the analytical approach of predicting the pharmaceutical property parameter of these inclusion compounds more exactly, but still desirable without comparison method in the prior art.
Summary of the invention
In order to adapt to the needs that cyclodextrin is used at drug world, fill up the blank of prior art, the purpose of this invention is to provide the interaction of a kind of medicine and cyclodextrin and the analysis and the characterizing method of pharmaceutical property thereof: utilize the method identification of Raman spectrum and the inclusion state or the inclusion degree of detection medicine/cyclodextrin system or pharmaceutical preparation Chinese traditional medicine.Solution is at key technical problems such as inclusion compound, the design of cyclodextrin pharmaceutical preparation, preparation process and storage Chinese traditional medicine inclusion state analysiss, and the pharmaceutical property parameter of prediction medicine is the stable and controlled proposition technological means of drug quality.
The technical scheme of invention is that the interaction of medicine and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof may further comprise the steps:
Collection of illustrative plates is measured: adopt Raman spectrometer or Fourier Raman spectrometer, sample to be measured is measured, obtain its Raman frequency spectrum or Fourier Raman frequency spectrum;
The selection of key band: ownership is also selected the absorption band or the absorption peak of inclusion compound collection of illustrative plates Chinese traditional medicine, select following one or more vibrational bands as feature with reference to bands of a spectrum:
Raman signatures is with reference to bands of a spectrum
Feature is with reference to the selection of bands of a spectrum, can by following steps or one of implement: by the molecular model of research medicine and cyclodextrin, the nuclear magnetic resoance spectrum research of medicine/cyclodextrin inclusion compound, determine that the clathration between drug molecule/cyclodextrin molecular is bigger to the influence of this group and bands of a spectrum thereof; The Raman spectrum of research medicine, cyclodextrin, pharmaceutical adjunct, system or preparation is determined in Raman spectrum, and the existence of cyclodextrin or other auxiliary materials of preparation can not disturbed this bands of a spectrum spectrum parameter (width, intensity, shift value, shape etc.) identification.
Collection:, collect the bands of a spectrum wave number, parameters such as spectral bandwidth, band intensity, bands of a spectrum shape by manual or employing mathematical analysis processing, function fitting method.As sample collection of illustrative plates and auxiliary material figure spectrum subtraction, baseline correction, deconvolute, nonlinear fitting, multiplet match, calculate derivative spectrum etc.
Result treatment
Qualitative analysis: the Raman collection of illustrative plates of the Raman collection of illustrative plates of specimen and control drug, contrast inclusion compound is compared, analyze correspondingly with reference to the peak or with reference to peak group's peak shape, peak width, peak intensity, position, basically identical judges whether inclusion; Estimate the variation of dissolving out capability parameter, solubility property parameter, permeance property parameter and the pharmacokinetic parameters of medicine.
The scope of application of such scheme of the present invention: be applicable to cyclodextrin and derivant thereof, as beta-schardinger dextrin-(β-CD), HP-(HP-β-CD), sulphur butyl ether-beta-schardinger dextrin-(SBE-β-CD); Be applicable to and comprise based on two components of medicine and cyclodextrin or multicomponent system (for example pbz polymer pharmaceutic adjuvant PVP, HPMC, PEG, various pharmaceutic adjuvants such as shitosan, PLA) and cyclodextrin pharmaceutical preparation.The pharmaceutical preparation system comprises various formulations such as liquid, solid, semisolid, as tablet, and emulsion, injection, micron or nano level drug delivery system.For example, the inclusion level determinations that contains the tablet of Nimodipine/hydroxypropyl cyclodextrin inclusion system.
The content range of system or preparation of traditional Chinese medicine is 0.1%～80%.
Further optimization of the present invention can increase following steps:
Quantitative test:
Criterion keying method: be provided with and the approaching standard control system of working sample condition, measure the Raman collection of illustrative plates respectively, from feature with reference to choosing best features absorption peak or characteristic absorption peak group the bands of a spectrum, with characteristic peak width, intensity, shift value, shape similarity coefficient is index, determine the optimum value of index, choose the ratio of the spectroscopic data of characteristic peak in sample and the reference substance collection of illustrative plates, carry out inclusion degree, stripping parameter, solubility parameter, permeability parameters, the pharmacokinetic parameters of calculation sample by following formula.
Inclusion degree or inclusion rate=(Γ s/ Γ r) * 100%,
Γ s is the spectral bandwidth of sample with reference to the peak, and Γ r is the spectral bandwidth of standard reference material with reference to the peak;
Inclusion degree or inclusion rate=(Rs/Rr) * 100%,
Rs is the similarity coefficient of sample with reference to the peak, and Rr is the similarity coefficient of standard reference material with reference to the peak, obtains by the corresponding function of match.
Inclusion degree or inclusion rate=(Δ fs/ Δ fr) * 100%
Δ fs is the shift value of sample with reference to the peak, and Δ fr is the shift value of standard reference material with reference to the peak;
Inclusion degree or inclusion rate=[(1-Is)/(1-Ir)] * 100%
Is is the peak strong coefficient of sample with reference to the peak, and Ir is the peak strong coefficient of standard reference material with reference to the peak;
Medicine stripping parameter PDs (t)=inclusion degree or inclusion rate * PDr (t)
PDs (t) is the stripping parameter of sample t time, and PDr (t) is the stripping parameter of standard reference material t time;
Medicine permeability parameters Papp/s=inclusion degree or inclusion rate * Papp/r,
Papp/s is the permeability parameters of sample, and Papp/r is the standard reference material permeability parameters;
The r of bioavilability parameter (AUC) s=inclusion degree or inclusion rate * (AUC)
AUC s is the bioavilability of sample, and AUC r is the standard reference material bioavilability.
The r of blood peak concentration of drug (Cmax) s=inclusion degree or inclusion rate * (Cmax)
(Cmax) s is the blood peak concentration of drug of sample, and (Cmax) r is the blood peak concentration of drug of standard reference material.
The r of peak time (Tmax) s=inclusion degree or inclusion rate * (Tmax)
(Tmax) s is the peak time of sample, and (Tmax) r is the peak time of standard reference material.
In actual analysis, can adopt excessive cyclodextrin (for example cyclodextrin: the mol ratio of medicine is 5: 1～10: 1) and adopt the higher preparation method of inclusion efficient (for example spray drying process etc.) preparation standard contrast inclusion compound; Measure the original collection of illustrative plates of this standard control inclusion compound, select the feature reference peak, collect spectrum parameters such as width, intensity, shift value, shape similarity coefficient; Selection changes the most responsive spectrum parameter index (can passing through relatively, the different spectrum parameters of the inclusion compound of different mol ratio obtain) for the inclusion degree, is optimum value with the spectrum parameter desired value of standard control thing feature reference peak, the inclusion rate of calculation sample.The stripping parameter of bioassay standard tester, permeability parameters, pharmacokinetic parameters, the stripping parameter of calculation sample, permeability parameters, pharmacokinetic parameters.According to optimum value, finally determine the pharmacy parameters such as relative inclusion degree, stripping parameter, permeability parameters, pharmacokinetic parameters of sample.
Above-mentioned quantitative test can be adopted working curve method or calibration curve method: serial reference substance (for example different host and guest's mol ratios etc. of choosing different inclusion degree, cyclodextrin for example: the mol ratio of medicine is 0.5: 1～8: 1), measure Raman spectrum respectively, select the feature reference peak, and select to change the most responsive spectrum index parameter (can passing through relatively, the different spectrum parameters of the inclusion compound of different mol ratio obtain) for the inclusion degree, make fixed reference feature peak spectrum parameter (width, intensity, shift value, the shape similarity coefficient) respectively with respect to the stripping parameter, solubility parameter, permeability parameters, the working curve of pharmacokinetic parameters or typical curve, by the spectrum parameter at mensuration or calculation sample fixed reference feature peak, obtain the stripping parameter of sample according to working curve or typical curve, solubility parameter, permeability parameters, pharmacokinetic parameters.
In actual analysis, can adopt Same Way (inclusion solwution method, polishing, freeze-drying, spray drying process etc.) preparation different mol ratio serial inclusion compound, measure original collection of illustrative plates, select the feature reference peak, obtain spectrum parameters such as width, intensity, shift value, shape similarity coefficient, measure its stripping parameter, permeability parameters, pharmacokinetic parameters respectively.Carry out regretional analysis, obtain the working curve of performance parameter and width, intensity, shift value, shape similarity coefficient, according to working curve and optimum value, finally determine the pharmacy parameters such as relative inclusion degree, stripping parameter, permeability parameters, pharmacokinetic parameters of sample.
Principle of work of the present invention
Cyclodextrin has outer hydrophilic, interior hydrophobic cage structure, whole molecule of lipophilicity object or lipophilic structure can be by hydrone and the so-called inclusion compounds of its formation (inclusion complexes) in the displacement CD cavity, and drug molecule or its group enter in the cup-shaped cavity of cyclodextrin and pass through various non-covalent interactions.Acting force between medicine and cyclodextrin molecular comprises Van der Waals force, electrostatic force, hydrogen bond action etc.Therefore, the free vibration of drug molecule or molecular radical is also hindered, and when molecular vibration followed dipole moment to change, the electromagnetic wave that vibration is produced changed the size that dipole moment changes when depending on vibration in fact.Because of intermolecular noncovalent interaction, cause the original peak of drug molecule to take place with the next item down or multinomial variation: significant change can take place in the shape at some peak, and the intensity at some peak can significantly descend, and some weak peaks will disappear; The obvious broadening of some peak width, some peak position is moved.Therefore, the shape of bands of a spectrum, intensity, peak width, position can have been reflected the various variations of mode of vibration from different perspectives, therefrom can understand effect degree change between medicine/cyclodextrin.
By research, the strong absorption band of some Ramans in the selection drug molecule is after being subjected to intermolecular noncovalent interaction, and the intensity of variation of various vibration modes depends on interaction degree between medicine/cyclodextrin.But expression inclusion in qualitative, quantitative ground has not reached the inclusion degree.Effect degree, medicine inclusion rate, medicament solubilization performance and permeance property are closely related between this variation and the medicine/cyclodextrin.
By research, the strong absorption band of some Ramans in the selection drug molecule can be avoided the interference of cyclodextrin and pharmaceutic adjuvant, can be used as with reference to the peak intuitively and analyzing.
The present invention has set up the simple recognition mode of a cover.In this cover pattern, select the feature Raman bands of a spectrum of medicine, effect degree in qualitative and quantitative analysis medicine/cyclodextrin systems such as the peak shape by key band, width, relative intensity, displacement between the component is discerned the inclusion state degree and the change trend of medicine/cyclodextrin system by the variation of bands of a spectrum relatively.The spectrum parameter (width, shift value, relative intensity, peak shape) of raman signatures bands of a spectrum and the quantitative relation of inclusion degree (inclusion rate), pharmaceutical property parameter (stripping parameter, solubility parameter, permeability parameters, pharmacokinetic parameters) have been set up.Thereby judge the inclusion state of medicine in preparation, the dissolving trend of prediction medicine and absorption, the transhipment behavior in the body.
Advantage of the present invention: have that specimen is untouchable, non-destructive, detection sensitivity height, the time is short, the sample aequum is little and sample need not characteristics such as preparation.Can under the condition of not destroying sample, discern the difference of low content medicine inclusion degree in the different systems.Be the inclusion trend and the inclusion rate state of quantitative detection inclusion system and pharmaceutical preparation Chinese traditional medicine, the effective ways of the pharmaceutical property of prediction medicine.
Description of drawings
Fig. 1 is the Raman spectrogram of Nimodipine and tablet thereof among the embodiment 1
Fig. 2 is the Raman spectrogram of brufen among the embodiment 2, HP-β-CD, inclusion compound.
Embodiment
(Nimodipine NM) is the second generation 1,4 dihydropyridine type calcium antagonists to Nimodipine.It is present wide clinical application cardiovascular and cerebrovascular medicine commonly used.This research adopts Nimodipine and hydroxypropyl cyclodextrin (behind the inclusion of HP-β-CD), form supramolecular system, and preparing tablet after adding corresponding auxiliary material, the employing Raman spectrum has carried out analyzing to the inclusion degree in the preparation, stripping parameter, solubility parameter, permeability parameters, pharmacokinetic parameters and has predicted and compare with experimental result.
The preparation of sample: adopt spray drying method for preparation Nimodipine/HP-β-CD supermolecule inclusion compound, Nimodipine/HP-β-CD mol ratio is 1: 3.This sample adds lactose, microcrystalline cellulose, superfine silica gel powder, dolomol, and adopting mixing, wet granulation, drying, compressing tablet to prepare specification is 10mg, and sheet heavily is the 200mg Nimodipine tablet.
The preparation of different mol ratio inclusion compound reference substance: adopt spray drying method for preparation Nimodipine/HP-beta-CD inclusion, adopt mixing, wet granulation, drying, compressing tablet preparation contrast tablet.Nimodipine/HP-β-CD mol ratio is 1: 1,1: 2,1: 3,1: 4,1: 5.
Raman spectrum test condition: laser excitation wavelength 514.5nm, laser power 10mw, resolution 2cm
-1, sweep limit 200～3200cm
-1Respectively reference substance and sample are measured.
The selection of characteristic peak and ownership: in the NM inclusion compound Raman spectrum a plurality of characteristic peaks are arranged, as 1696cm
-1, 1642cm
-1, 1581cm
-1, 1491cm
-1, 1345cm
-1, 1198cm
-1, 1132cm
-1, 1095cm
-1, 1004cm
-1Deng; A plurality of characteristic peaks are arranged, as 1458cm in the Raman spectrum of HP-
-1, 1390cm
-1, 1330cm
-1, 1255cm
-1, 1124cm
-1, 927cm
-1, 854cm
-1Characteristic peak in the Raman spectrum of other auxiliary material is as 1486cm
-1, 1388cm
-1, 1102cm
-1, 444cm
-1, 387cm
-1The 1696cm of NM in the Raman spectrum of tablet
-1, 1642cm
-1, 1581cm
-1, 1491cm
-1, 1345cm
-1, 1004cm
-1Can know explanation Deng still, especially 1696cm
-1, 1642cm
-1, 1581cm
-1Not disturbed by auxiliary material; Prove all that by HNMR, CNMR spectrum and appliance computer assistant chemical software (Computer-Aided Chemistry) simulation of measuring NM, inclusion compound the easiest cyclodextrin cavity that enters of carbonyl in the molecule, its bands of a spectrum can be used as feature with reference to peak indication inclusion degree.By relatively finding 1642cm
-1Bands of a spectrum be strong peak, the variation sensitivity of parameter, select as quantitative test with reference to the peak, referring to accompanying drawing 1.
NM key band and ownership
Collection: use origin software with in the different collection of illustrative plates of Lorentzian function match with reference to peak 1642cm
-1Peak shape; Mensuration is with reference to peak 1642cm
-1Peak width.
Interpretation of result:
Qualitative analysis 1: the Raman bands of a spectrum 1696cm of Nimodipine
-1Obviously there is the Raman bands of a spectrum 1696cm in 1: 2 inclusion reference substance
-1Disappear, these bands of a spectrum disappear in the Raman spectrum of inclusion compound and tablet samples, and Nimodipine and HP-β-CD inclusion is described in inclusion compound and the tablet samples.
Qualitative analysis 2: the Raman bands of a spectrum 1642cm of Nimodipine
-1Peak width is 80cm
-1, the Raman bands of a spectrum 1642cm in 1: 3 inclusion reference substance
-1Peak width is 124cm
-1, the bands of a spectrum 1642cm of inclusion compound and tablet samples
-1Peak width is respectively 122cm
-1, 127cm
-1, Nimodipine and HP-β-CD inclusion is described in inclusion compound and the tablet samples.
Quantitative test 1: the situation of change of the vibrational band at each character pair peak relatively, find carbonylic stretching vibration bands of a spectrum 1642cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Be strong absorption peak in the collection of illustrative plates, its shape, peak width, Strength Changes are the most remarkable, and as the reference peak, the peak type is with the match of Lorentzian function with this peak, and the peak shape similarity coefficient of inclusion compound sample is 0.736; The peak shape similarity coefficient of tablet samples is 0.732.The peak shape similarity coefficient increases and significantly reduces with HP-β-CD content in the collection of illustrative plates of different mol ratio inclusion compound reference substance, the inclusion degree increases in the explanation system, mol ratio is that the inclusion compound peak shape similarity coefficient of 1: 4 and 1: 5 is very approaching, be respectively 0.704 and 0.702, illustrate that to reach inclusion saturated.With peak shape similarity coefficient (R) is index, with the shape similarity coefficient (0.702) of 1: 5 inclusion compound as optimum value, being calculated as follows the relative inclusion degree or the inclusion rate that draw the inclusion compound sample is 88.6%, and the relative inclusion degree or the inclusion rate of tablet samples are 89.9%.
Inclusion degree or inclusion rate (complexation efficiency)=(1-Rs/1-Rr) * 100%Rs is the similarity coefficient of sample with reference to the peak, and Rr is the similarity coefficient of standard reference material with reference to the peak.
Quantitative test 2: the situation of change of the vibrational band at each character pair peak relatively, find carbonylic stretching vibration bands of a spectrum 1642cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Be strong absorption peak in the collection of illustrative plates, its shape, peak width, Strength Changes are the most remarkable, and peak width as the reference peak, is measured in this peak, and the peak width of inclusion compound sample is 94cm
-1The peak width of tablet samples is 96cm
-1With the peak width is index, with the peak width (109cm of 1: 5 inclusion compound
-1) as optimum value, being calculated as follows the relative inclusion degree or the inclusion rate that draw the inclusion compound sample is 85.7%.The relative inclusion degree or the inclusion rate of tablet samples are 88.1%.
Inclusion degree or inclusion rate (complexation efficiency)=(Γ s/ Γ r) * 100%
Γ s is the spectral bandwidth of sample with reference to the peak, and Γ r is the spectral bandwidth of standard reference material with reference to the peak.
Quantitative test 3: select feature with reference to peak 1642cm
-1Measure in the Raman spectrum of serial inclusion tester feature with reference to peak 1642cm
-1Peak width.Respectively with the stripping parameter PD of inclusion tester
5After carrying out regretional analysis, set up stripping parameter-peak width working curve, by the stripping parameter of working curve quantitative Analysis medicine, the stripping parameter of prediction medicine.
Select feature with reference to peak 1642cm
-1Measure in the Raman spectrum of serial inclusion tester feature with reference to peak 1642cm
-1Peak width.Respectively with respective tablets stripping parameter PD
15After carrying out regretional analysis, set up stripping parameter-peak width working curve (r=0.966), by the stripping parameter of working curve quantitative Analysis medicine, the stripping parameter of prediction medicine.
Stripping parameter-peak width working curve
According to feature in the inclusion compound sample collection of illustrative plates with reference to peak 1642cm
-1Peak width be 117cm
-1, calculate PD according to working curve
5=38.9%, the practical measurement data are 35.7%.
According to feature in the tablet samples collection of illustrative plates with reference to peak 1642cm
-1Peak width be 120cm
-1, calculate PD according to working curve
15=41.4%, the practical measurement data are 44.7%.
Quantitative test 4: select feature with reference to peak 1642cm
-1Measure in the Raman spectrum of different inclusion compounds feature with reference to peak 1642cm
-1Peak width.Set up corresponding C aco2 Premeabilisation of cells model, different inclusion compound reference substances are measured corresponding permeability parameters (Papp) on saturated model, after peak width and permeability parameters (Papp) carried out regretional analysis, set up permeability parameters-peak width working curve (r=0.899), by the permeability parameters of working curve quantitative Analysis medicine.The permeability parameters of prediction inclusion compound, tablet samples Chinese traditional medicine.
Infiltration coefficient-peak width working curve
According to feature in the inclusion compound sample collection of illustrative plates with reference to peak 1642cm
-1Peak width be 117cm
-1, calculate Papp=7.32 * 10 according to working curve
-6, the practical measurement data are P=7.26 * 10
-6
According to feature in the tablet samples collection of illustrative plates with reference to peak 1642cm
-1Peak width be 120cm
-1, calculate Papp=7.62 * 10 according to working curve
-6, the practical measurement data are Papp=7.66 * 10
-6
Cellular incubation Caco-2 cell inoculation is in conventional culture flask, and nutrient culture media is DMEM, contains 1% nonessential amino acid, 10% hyclone, and cellular incubation is (37 ℃, 5%CO2, relative humidity 90%) in the CO2 incubator.When cell grow to cover the bottle end 80%～90% the time, trypsinization, and be inoculated on the polycarbonate membrane of Transwell (0.5mL/ hole), after adding nutrient solution 1.5mL. inoculation 24h, the cavity of resorption of Transwell changes nutrient solution. and next a week is changed liquid every other day, change liquid every day after one week, day stand-by surplus cultivating 20.The cell monolayer resistance measurement is incubated at Caco-2 among the Transwell with preceding method, measures cell monolayer resistance (TEER) value every day, and TEER reaches 300 Ω/cm
2Available dried penetrating experiment.Carefully wash with PBS to remove the metabolin that is attached on cell surface.Add medicine in the AP pond, the DMEM nutrient solution that adds serum-free in the BL pond, hatch in 37 ℃ of incubators. respectively at 15,45,60,120,150min is from BL pond sampling 0.5mL, and the serum-free DMEM nutrient solution of adding equivalent immediately is in the BL pond, with HPLC working sample liquid Chinese traditional medicine concentration, and by following formula calculating penetrating amount Δ Q of accumulative total and apparent infiltration coefficient Papp.
ΔQ＝CnV+∑CiΔV(i＝1，2…，n-1)
Wherein, Δ Q is the penetrating amount of accumulative total, and Cn is the drug concentration of measuring each time, and V is a BL side liquid volume, and Δ V is a sample volume each time.
Papp＝(dQ/dt)/(A·C
0)
Wherein dQ/dt is unit interval drug transport amount (μ mols
-1), A is the area (cm of carrier film
2), C
0Initial concentration (μ molL for medicine
-1).
Quantitative test 5: select feature with reference to peak 1642cm
-1, peak type (1600-1680cm
-1) with the match of Lorentzian function, obtain in the Raman spectrum of different tablet testers feature with reference to peak 1642cm
-1The peak shape similarity coefficient.And measure the relative bioavailability of different tablet testers, after carrying out regretional analysis by peak shape similarity coefficient and tablet bioavilability parameter A UC, set up bioavilability-peak shape similarity coefficient working curve (r=0.947), by the AUC of working curve quantitative Analysis medicine.The prediction bioavailability of medicament.
The mensuration of biological blood concentration and the estimation of pharmacokinetic parameters
Domesticated dog, body weight 10 ± 2kg, male and female are not limit, and are provided by China Medicine University's Experimental Animal Center.Be divided into some groups at random, 7 every group.
Administering mode: adopt the experimental design of single dose dual crossing.The every wheel 1 week of interval between the administration.With medical forceps medicine is positioned over the pharyngeal of domesticated dog during administration, gavages distilled water 50ml again after domesticated dog swallows naturally.Embrace domesticated dog by the keeper during blood sampling, get blood from domesticated dog four limbs superficial vein.
Blood sampling time: take medicine preceding and take medicine about back blood sampling in 0.5,1,1.5,2,3,4,5,6 hour 2ml in the heparinize test tube, centrifuging and taking blood plasma place-20 ℃ freezing immediately, the HPLC method is measured blood concentration.
The result calculates: adopt the blood concentration-time curve of every group of sample of mean value drafting as a result, and carry out pharmacokinetic parameters and calculate.Can directly obtain blood peak concentration of drug (Cmax) and peak time (Tmax) by experimental data.
Area under the concentration time curve (AUC 0-∞): AUC 0-∞=AUC
t+ C
t/ λ
ZAUC in the formula
tCalculate with trapezoidal rule, t is last minute point; λ
ZBe terminal phase rate constant, promptly the end to blood medicine log logarithm concentration-time data carries out straight-line regression, and the absolute value of gained slope multiply by 2.303 and is λ
ZC
tBlood concentration for last time point.
Bioavilability-peak shape similarity coefficient working curve
According to feature in the tablet samples collection of illustrative plates with reference to peak 1642cm
-1Similarity coefficient be 0.837, calculate AUC=1155ng h/ml according to working curve, the practical measurement data are 1108ng h/ml.
According to feature in the tablet samples collection of illustrative plates with reference to peak 1642cm
-1Similarity coefficient be 0.837, calculate C according to working curve
Max=236.8ng/ml, the practical measurement data are 228.7ng/ml.
According to feature in the tablet samples collection of illustrative plates with reference to peak 1642cm
-1Similarity coefficient be 0.837, calculate T according to working curve
Max=45.7min, the practical measurement data are 43.9min.
Embodiment 2, the Raman spectrum characterization research of the interaction of brufen and HP-and pharmaceutical property thereof
(ibuprofen is that present clinical efficacy is definite, bad reaction is little, safe alkyl propionic acid class antipyretic-antalgic anti-inflammatory agent is in wide clinical application bf) to brufen.But because poorly water-soluble has influenced bioavilability, existing both at home and abroad many pieces of bibliographical informations adopt water soluble cyclodextrin derivant to prepare supermolecule to change
Kind water-soluble and bioavilability.This research adopts brufen and HP-(behind HP-β-CD), the sulfobutyl ether cyclodextrin SBE-β-CDBao He, form supramolecular system, and preparing capsule after adding corresponding auxiliary material, the employing Raman spectrum has carried out analyzing to the inclusion degree in the preparation, stripping parameter, solubility parameter, permeability parameters, bioavilability parameter and has predicted and compare with experimental result.
The preparation of sample: adopt polishing to prepare brufen/HP-β-CD supermolecule inclusion compound.Brufen/HP-β-CD mol ratio is 1: 1.This sample adds lactose, microcrystalline cellulose, superfine silica gel powder, and the preparation specification is 10mg, heavily is the agent of 200mg ibuprofen capsule.
The preparation of different mol ratio inclusion compound reference substance: adopt polishing to prepare brufen/HP-β-CD supermolecule inclusion compound and corresponding capsule respectively.Brufen/HP-β-CD mol ratio is 1: 0.5,1: 0.8,1: 1,1: 1.5,1: 2.
Raman spectrum test condition: laser excitation wavelength 785nm, laser power 10mw, resolution 2cm
-1, sweep limit 200～3200cm
-1Respectively reference substance and sample are measured.
The selection of characteristic peak and ownership: the situation of change of the vibrational band at each character pair peak relatively, find carbonylic stretching vibration bands of a spectrum 1606cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Be strong absorption peak in the collection of illustrative plates, displacement and change of shape have taken place, its change of shape is the most remarkable, and not disturbed by cyclodextrin and auxiliary material, prove all that by HNMR, CNMR spectrum and appliance computer assistant chemical software (Computer-Aided Chemistry) simulation of measuring brufen, inclusion compound the easiest cyclodextrin cavity that enters of carbonyl in the molecule, its bands of a spectrum can be used as feature with reference to peak indication inclusion degree.Referring to accompanying drawing 2.
Collection: bands of a spectrum 1606cm in the brufen Raman spectrum
-1Peak type (1580-1620cm
-1) with Lorentzian, the match of Gaussian function, be index with the match similarity coefficient of sample.
Interpretation of result:
Qualitative analysis: the Raman bands of a spectrum 1606cm of brufen
-1Peak type (1580-1620cm
-1) with the match of Lorentzian function, similarity coefficient is 0.964; Similarity coefficient is that the similarity coefficient of 0.782,1: 1 inclusion compound sample is 0.767 after this Raman bands of a spectrum match in 1: 1 inclusion reference substance, and the similarity coefficient of capsule sample is 0.759, brufen and HP-β-CD inclusion in the interpret sample.
Quantitative test 1: the peak shape (1590-1630cm of inclusion compound sample
-1) similarity coefficient is 0.744, the peak shape (1590-1630cm of capsule sample
-1) similarity coefficient is 0.759, be index with peak shape similarity coefficient (R) respectively, with 1: 2 the contrast inclusion compound peak shape similarity coefficient (0.710) as optimum value, be calculated as follows draw the inclusion compound sample comprise degree relatively or inclusion rate is 88.3%, the relative inclusion degree or the inclusion rate of tablet samples are 83.1%.
Inclusion degree or inclusion rate (complexation efficiency)=(1-Rs/1-Rr) * 100%Rs is the similarity coefficient of sample with reference to the peak, and Rr is the similarity coefficient of standard reference material with reference to the peak.
Quantitative test 2: select feature with reference to peak 1606cm
-1Measure in the Raman spectrum of serial inclusion tester feature with reference to peak 1606cm
-1The peak shape similarity coefficient.After carrying out regretional analysis with inclusion tester stripping parameter PD10 respectively, set up stripping parameter-peak shape similarity coefficient working curve (r=0.988), by the stripping parameter of working curve quantitative Analysis medicine, the stripping parameter of prediction medicine.
Stripping experiment precision takes by weighing an amount of sample (being equivalent to the 10mg brufen), according to the Pharmacopoeia of the People's Republic of China two appendix XC slurry method operations in 2000, with the 900mL redistilled water is dissolution medium, 37 ℃, rotating speed 50rpm, respectively at stipulated time sampling 8mL, replenish the equivalent medium simultaneously, 0.45 μ m miillpore filter filters, with the redistilled water is blank, according to spectrophotometric method (2000 editions two appendix IV A of Pharmacopoeia of People's Republic of China), measure absorbance at the 225nm place, calculate cumulative leaching rate %.
Select feature with reference to peak 1606cm
-1Measure in the Raman spectrum of serial inclusion tester feature with reference to peak 1606cm
-1The peak shape similarity coefficient.After carrying out regretional analysis with corresponding capsule stripping parameter PD30, set up stripping parameter-peak shape similarity coefficient working curve (r=0.951), by the stripping parameter of working curve quantitative Analysis medicine, the stripping parameter of prediction medicine.
Stripping parameter-similarity coefficient working curve
According to feature in the inclusion compound sample collection of illustrative plates with reference to peak 1606cm
-1Similarity coefficient be 0.753, calculate PD5=38.9% according to working curve, the practical measurement data are 35.7%.
Following table is represented capsule stripping parameter PD
30The feature of-coefficient of variation working curve:
Stripping parameter-coefficient of variation working curve
Per sample in the collection of illustrative plates feature with reference to peak 1606cm
-1Similarity coefficient be 0.753, calculate PD according to working curve
30=83.4%, the practical measurement data are 85.7%.
Quantitative test 3: select feature with reference to peak 1606cm
-1Measure in the Raman spectrum of different inclusion compounds, capsule tester feature with reference to peak 1606cm
-1The peak shape similarity coefficient.Set up corresponding saturated model, different inclusion compounds are measured corresponding permeability parameters (Papp) on saturated model, after carrying out regretional analysis with corresponding permeability parameters (Papp) respectively, set up permeability parameters-similarity coefficient working curve (r=0.919), by the permeability parameters of working curve quantitative Analysis medicine.The permeability parameters of prediction inclusion compound, capsule sample Chinese traditional medicine.
Cell traffic experiment Caco-2 cell inoculation is in conventional culture flask, and nutrient culture media is DMEM, contains 1% nonessential amino acid, 10% hyclone, and cellular incubation is (37 ℃, 5%CO2, relative humidity 90%) in the CO2 incubator.When cell grow to cover the bottle end 80%～90% the time, trypsinization, and being inoculated on the polycarbonate membrane of Transwell (0.5mL/ hole) adds nutrient solution 1.5mL at the cavity of resorption of Transwell.Change nutrient solution behind the inoculation 24h.Next a week is changed liquid every other day, and change liquid every day after the week, and is day stand-by surplus cultivating 20.The cell monolayer resistance measurement is incubated at Caco-2 among the Transwell with preceding method, measures cell monolayer resistance (TEER) value every day, and TEER reaches 300 Ω/cm
2Available dried penetrating experiment.Carefully wash with PBS to remove the metabolin that is attached on cell surface.Add medicine in the AP pond, add the DMEM nutrient solution of serum-free in the BL pond, hatch in 37 ℃ of incubators.Respectively at 15,45,60,120,150min is from BL pond sampling 0.5mL, and the serum-free DMEM nutrient solution of adding equivalent immediately is in the BL pond, with HPLC working sample liquid Chinese traditional medicine concentration, and by following formula calculating penetrating amount Δ Q of accumulative total and apparent infiltration coefficient Papp.
ΔQ＝CnV+∑CiΔV(i＝1，2…，n-1)
Wherein, Δ Q is the penetrating amount of accumulative total, and Cn is the drug concentration of measuring each time, and V is a BL side liquid volume, and Δ V is a sample volume each time.
Papp＝(dQ/dt)/(A·C
0)
Wherein dQ/dt is unit interval drug transport amount (μ molL
-1), A is the area (cm of carrier film
2), be initial concentration (the μ molL of medicine
-1).
Infiltration coefficient one similarity coefficient working curve
Per sample in the collection of illustrative plates feature with reference to peak 1606cm
-1Similarity coefficient be 0.753, calculate Papp=21.4 according to working curve, the practical measurement data are 22.6.
Quantitative test 4: select feature with reference to peak 1606cm
-1, the peak type is with the match of Lorentzian function, and feature is with reference to peak 1606cm in the Raman spectrum of acquisition capsule tester
-1The peak shape similarity coefficient.And measure the relative bioavailability of different capsule testers, after carrying out regretional analysis by peak shape similarity coefficient and capsule bioavilability parameter A UC, set up bioavilability-peak shape similarity coefficient working curve (r=0.875), by the AUC of working curve quantitative Analysis medicine.The prediction bioavailability of medicament.
Bioavilability-peak shape similarity coefficient working curve
Please refer to subordinate list: the feature of tablet bioavilability AUC-peak shape similarity coefficient R working curve.
According to feature in the tablet samples collection of illustrative plates with reference to peak 1642cm
-1Similarity coefficient be 0.753, calculate AUC=1255.6ng h/ml according to working curve, the practical measurement data are 1262.1ng h/ml.
Embodiment 3, the interaction of Cefditoren pivoxil Cephalosporins and sulphur butyl ether-beta-schardinger dextrin-and the Raman spectrum characterization research of pharmaceutical property thereof
Cefditoren pivoxil Cephalosporins (Cefditoren Pivoxil) be the 4th generation the antibacterial cephalosporin element, gram-positive bacteria and negative bacterium had extensive antimicrobial spectrum, especially to staphylococcus, the gram-positive bacterias such as streptococcus that comprise streptococcus pneumonia, anaerobions such as Gram-negative bacteria such as Escherichia coli, catarrh Branhamella catarrhalis, klebsiella, Proteus, haemophilus influenzae and Peptostreptococcus, propionibacterium acnes, Bacteroides show very strong antimicrbial power.Cefditoren pivoxil Cephalosporins and sulphur butyl ether-beta-schardinger dextrin-are adopted in this research
(behind the inclusion of SBE-β-CD), form supramolecular system, and prepare granule after adding corresponding auxiliary material, adopt Raman spectrum that the inclusion degree in the preparation, stripping parameter have been carried out analyzing and prediction and compare with experimental result.
The preparation of sample: adopt polishing to prepare Cefditoren pivoxil Cephalosporins/SBE-β-CD supermolecule inclusion compound.Cefditoren pivoxil Cephalosporins/SBE-β-CD mol ratio is 1: 1.This sample adds lactose, dextrin etc., the granule of preparation 100mg specification.
The preparation of different mol ratio inclusion compound reference substance: adopt polishing to prepare Cefditoren pivoxil Cephalosporins/HP-β-CD supermolecule inclusion compound reference substance respectively.Cefditoren pivoxil Cephalosporins/SBE-β-CD mol ratio is 1: 0.5,1: 0.8,1: 1,1: 1.5,1: 2.
Raman spectrum test condition: laser excitation wavelength 514.5nm, laser power 10mw, resolution 2cm
-1, sweep limit 200～3200cm
-1Respectively reference substance and sample are measured.
The selection of characteristic peak and ownership: 1600cm in the Cefditoren pivoxil Cephalosporins Raman spectrum
-1Characteristic peak belongs to the vibration of conjugated double bond, 1300cm
-1Characteristic peak belongs to the vibration of ester group.By to 1600cm in the ME1207 ester Raman spectrum
-1, 1300cm
-1The mathematics of characteristic peak deconvolute analysis, the match of Lorentzian function are with 1600cm
-1The bands of a spectrum at place are divided into 1620cm
-1, 1590cm
-1, 1570cm
-1, 1564cm
-1Four inferior peaks, 1300cm
-1The bands of a spectrum at place are divided into 1340cm
-1, 1311cm
-1, 1290cm
-1Three inferior peaks.
Collection: the situation of change of the vibrational band at each character pair peak relatively, find vibrational band 1590cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Be strong absorption peak in the collection of illustrative plates, its Strength Changes is the most remarkable, and not disturbed by cyclodextrin and auxiliary material, with this peak as the reference peak.By multianalysis, prove that clathration is to 1500cm in the ME1207 ester collection of illustrative plates
-1The intensity at peak, place does not have influence.Calculate 1590cm in the different sample spectra
-1The intensity and the corresponding 1500cm at peak, place
-1The ratio of the intensity at peak, place as the strong variation factor in peak (Ip), is that index is analyzed with the strong variation factor in peak.
Result data is handled:
Qualitative analysis 1: 1590cm in the Cefditoren pivoxil Cephalosporins Raman spectrum after mathematics manipulation
-1, 1290cm
-1Intensity obviously descends in the Raman spectrum of inclusion compound sample and granule sample, Cefditoren pivoxil Cephalosporins and SBE-β-CD inclusion in the interpret sample.
Quantitative test 1: the strong variation factor in the peak of inclusion compound sample is 0.364, the strong variation factor in the peak of granule sample is 0.359, with 1: 2 the contrast inclusion compound the strong variation factor in peak (0.275) as optimum value, being calculated as follows the relative inclusion degree or the inclusion rate that draw the inclusion compound sample is 87.7%, and the relative inclusion degree or the inclusion rate of granule sample are 88.4%.
Inclusion degree or inclusion rate (complexation efficiency)=(1-Is/1-Ir) * 100%Rs is the peak strong variation factor of sample with reference to the peak, and Rr is the peak strong variation factor of standard reference material with reference to the peak.Quantitative test 2: measure in the Raman spectrum of serial inclusion tester feature with reference to peak 1590cm
-1The strong variation factor in peak (Ip).After carrying out regretional analysis with inclusion tester stripping parameter PD5 and granule stripping parameter PD30 respectively, set up the strong variation factor in stripping parameter PD5-peak (Ip) working curve (r=0.985), the number strong variation factor in PD30-peak (Ip) working curve (r=0.955), by the stripping parameter of working curve quantitative Analysis medicine, the stripping parameter of prediction medicine.
The strong variation factor in stripping parameter-peak (Ip) working curve
According to feature in the inclusion compound sample collection of illustrative plates with reference to peak 1590cm
-1Peak strong variation factor (Ip) be 0.364, calculate PD5=72.6% according to working curve, the practical measurement data are 73.8%.
According to feature in the granule sample collection of illustrative plates with reference to peak 1590cm
-1Peak strong variation factor (Ip) be 0.355, calculate PD according to working curve
30=73.4%, the practical measurement data are 75.7%.
Embodiment 4, the Raman spectrum characterization research of the interaction of totokaine and HP-and pharmaceutical property thereof
Totokaine (Tetracaine) is the arcotic of wide clinical application.This research adopt totokaine and HP-(behind the inclusion of HP-β-CD), form supramolecular system, and add preparation powder pin behind the corresponding auxiliary material, adopt Raman spectrum that the inclusion degree in the preparation, stripping parameter have been carried out analyzing and predict and with reality
Testing the result compares.
The preparation of sample: adopt freeze-drying to prepare totokaine/HP-β-CD supermolecule inclusion compound.Totokaine/HP-β-CD mol ratio is 1: 1.Totokaine, HP-β-CD, sweet mellow wine adopts batching, filtration, freeze-drying to prepare the powder pin that specification is 50mg.
The preparation of different mol ratio inclusion compound reference substance: adopt freeze-drying to prepare totokaine/HP-β-CD supermolecule inclusion compound and corresponding totokaine respectively.Totokaine/HP-β-CD mol ratio is 1: 1,1: 1.5,1: 2,1: 2.5,1: 3.
Raman spectrum test condition: laser excitation wavelength 514.5nm, laser power 10mw, resolution 2cm
-1, sweep limit 200～3200cm
-1Respectively reference substance and sample are measured.
The selection of characteristic peak and ownership: characteristic peak belongs to the vibration of aromatic rings, 1690cm in the totokaine Raman spectrum
-1Characteristic peak belongs to the vibration of ester group.The situation of change of the vibrational band at each character pair peak is relatively found vibrational band 1601cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Be strong absorption peak in the collection of illustrative plates, its change in displacement is the most remarkable, and not disturbed by cyclodextrin and auxiliary material, with this peak as the reference peak.
Collection: calculate 1601cm in the different sample spectra
-1The shift value (Δ f) at peak, place.Index as a comparison.
Result data is handled:
Qualitative analysis 1: 1601cm in the totokaine Raman spectrum
-1, 1690cm
-1Characteristic peak is 1601cm in the Raman spectrum of inclusion compound sample and powder sample needle
-1Move to 1616cm to high wave number
-1, 1690cm
-1The obvious broadening of characteristic peak, totokaine and HP-β-CD inclusion in the interpret sample.
Quantitative test 1: calculate 1601cm in the different sample spectra
-1The shift value at peak, place.Inclusion compound sample displacement value is 12.4cm
-1, the shift value of powder sample needle is 12.9cm
-1, (Δ f) is index with shift value, with the shift value (15.7cm of 1: 3 contrast inclusion compound
-1) as optimum value, being calculated as follows the relative inclusion degree or the inclusion rate that draw the inclusion compound sample is 82.7%, relative inclusion degree of powder sample needle or inclusion rate are 86.0%.
Inclusion degree or inclusion rate (complexation efficiency)=(Δ fs/ Δ fr) * 100% Δ fs is the shift value of sample with reference to the peak, and Δ fr is the shift value of standard reference material with reference to the peak.
Quantitative test 2: select feature with reference to peak 1601cm
-1, measure in the Raman spectrum of serial inclusion tester feature with reference to peak 1601cm
-1Shift value (Δ f).After carrying out regretional analysis with inclusion tester stripping parameter PD1 respectively, set up inclusion compound stripping parameter PD1-peak shift value (Δ f) working curve (r=0.992), after carrying out regretional analysis with corresponding powder pin stripping parameter PD1 respectively, set up powder pin stripping parameter PD1-peak shift value (Δ f) working curve (r=0.979), by the stripping parameter of working curve quantitative Analysis, prediction medicine.
Stripping parameter-peak shift value (Δ f) working curve
Is 12.4cm according to feature in the inclusion compound sample collection of illustrative plates with reference to the peak shift value (Δ f) at peak
-1, calculate PD1=68.4% according to working curve, the practical measurement data are 66.7%.
Is 12.9cm according to feature in the powder sample needle collection of illustrative plates with reference to the peak shift value (Δ f) at peak
-1, calculate PD according to working curve
1=70.4%, the practical measurement data are 71.7%.
Embodiment 5, the Raman spectrum characterization research of the interaction of amantadine hydrochloride and HP-and pharmaceutical property thereof
Amantadine hydrochloride (Amantadine hydrochloride) is used for prevention and the treatment caused respiratory tract infection of influenza virus and Antiparkinsonian and parkinsonism.This research adopts amantadine hydrochloride and HP-(behind the inclusion of HP-β-CD), form supramolecular system, and preparing injection after adding corresponding auxiliary material, the employing Raman spectrum has carried out analyzing to the inclusion degree in the preparation, inclusion rate and has predicted and compare with experimental result.
The preparation of sample: adopt saturated solution method to prepare amantadine hydrochloride/HP-β-CD supermolecule inclusion compound.Amantadine hydrochloride/HP-β-CD mol ratio is 1: 2.Prepare the injection that specification is 100mg by batching, filtration, embedding, sterilization.
The preparation of different mol ratio inclusion compound reference substance: adopt saturated solution method to prepare amantadine hydrochloride/HP-beta-CD inclusion solution respectively.Amantadine hydrochloride/HP-β-CD mol ratio is 1: 1,1: 1.5,1: 2,1: 3,1: 4.
Raman spectrum test condition: laser excitation wavelength 514.5nm, laser power 10mw, resolution 2cm
-1, sweep limit 200～3200cm
-1Respectively reference substance and sample are measured.
The selection of characteristic peak and ownership: strong absorption band 720,777 in the amantadine hydrochloride aqueous solution Raman spectrum, 1218cm
-1Characteristic peak belongs to the C-C stretching vibration, CH2 rocking vibration, CH flexural vibrations.
Collection: the situation of change of the vibrational band at each character pair peak relatively, find 777cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Bands of a spectrum are strong absorption peak in the collection of illustrative plates, and its change in displacement is the most remarkable, and not disturbed by cyclodextrin and auxiliary material, with this peak as the reference peak.Calculate 777cm in the different sample spectra
-1The shift value at peak, place.
Result data is handled:
Qualitative analysis 1: 777cm in the amantadine hydrochloride Raman spectrum
-1Characteristic peak these bands of a spectrum in the Raman spectrum of injection sample move to 793cm to high wave number
-1720cm
-1Characteristic peak these bands of a spectrum in the Raman spectrum of injection sample move to 731cm to high wave number
-11218cm
-1Characteristic peak these bands of a spectrum in the Raman spectrum of injection sample move to 1206cm to lower wave number
-1Clathration has taken place in amantadine hydrochloride and HP-β-CD in the interpret sample.
Quantitative test 1: the shift value of injection sample is 16cm
-1, to contrast the shift value (22cm of inclusion compound (the inclusion degree is the highest) at 1: 4
-1) as optimum value, being calculated as follows the relative inclusion degree or the inclusion rate that draw the injection sample is 72.7%.
Inclusion degree or inclusion rate (complexation efficiency)=(Δ fs/ Δ fr) * 100% Δ fs is the shift value of sample with reference to the peak, and Δ fr is the shift value of standard reference material with reference to the peak.
Embodiment 6, the Raman spectrum characterization research of the interaction of Chlorhexidine and HP-and pharmaceutical property thereof
Chlorhexidine (Chlorhexidine) is surperficial quiet property formulation germifuge, and is all effective to gram positive bacteria and gram-negative bacteria.Gargle is used for stomatitis; Suppository is used for bacterial vaginitis, hemorrhoid.This research adopts Chlorhexidine and HP-(behind the inclusion of HP-β-CD), form supramolecular system, and preparing suppository or GARG after adding corresponding auxiliary material, the employing Raman spectrum has carried out analyzing to the inclusion degree in the preparation, stripping parameter, solubility parameter, permeability parameters and has predicted and compare with experimental result.
The preparation of sample: adopt spray drying method for preparation Chlorhexidine/HP-β-CD supermolecule inclusion compound.Chlorhexidine/HP-β-CD mol ratio is 1: 1.This sample adds auxiliary materials such as cocoa butter, paraffin, and the preparation specification is 20 milligrams of suppositorys.Adopt technologies such as batching, filtration, can to prepare 0.2% Chlorhexidine gargle.
The preparation of different mol ratio inclusion compound reference substance: adopt spray drying method for preparation Chlorhexidine/HP-β-CD supermolecule inclusion compound respectively.Chlorhexidine/HP-β-CD mol ratio is 1: 0.5,1: 0.8,1: 1,1: 1.5,1: 2.
Raman spectrum test condition: laser excitation wavelength 514.5nm, laser power 10mw, resolution 2cm
-1, sweep limit 200～3200cm
-1Respectively reference substance and sample are measured.
The selection of characteristic peak and ownership: 1564cm in the Chlorhexidine Raman spectrum
-1Characteristic peak belongs to the vibration of conjugated double bond.Be strong absorption peak in the collection of illustrative plates, and not disturbed by cyclodextrin and auxiliary material, with this peak as the reference peak.
Collection: the situation of change of the vibrational band at each character pair peak relatively, find bands of a spectrum 1564cm in the collection of illustrative plates of inclusion compound of different inclusion degree
-1Change in displacement the most remarkable, calculate 1564cm in the different sample spectra
-1The shift value at peak, place.
Result data is handled:
Qualitative analysis 1: 1564cm in the Chlorhexidine Raman spectrum
-1Characteristic peak in the Raman spectrum of inclusion compound sample and suppository, gargle sample to high wave number move to 1614,1611,1609cm
-1Chlorhexidine and HP-β-CD inclusion in the interpret sample.
Quantitative test 1: calculate 1564cm in the different sample spectra
-1The shift value at peak, place.The shift value of inclusion compound sample is 50cm
-1, the shift value of suppository sample is 47cm
-1, the shift value of gargle sample is 45cm
-1, to contrast the shift value (57cm of inclusion compound (the inclusion degree is the highest) at 1: 2
-1) as optimum value, being calculated as follows and drawing the inclusion degree or inclusion rate is respectively: inclusion compound sample 87.7%, suppository sample are 82.5, the gargle sample is 78.9%.
Inclusion degree or inclusion rate (complexation efficiency)=(Δ fs/ Δ fr) * 100%.
Claims (5)
1, the Raman spectrum analysis method of the interaction of a kind of medicine and cyclodextrin and pharmaceutical property thereof may further comprise the steps:
Collection of illustrative plates is measured: adopt Raman spectrometer or Fourier Raman spectrometer, medicine to be measured and cyclodextrin inclusion compound are measured, obtain its Raman spectrum or Fourier Raman spectrum;
The selection of key band: select and the absorption band or the absorption peak of ownership inclusion compound collection of illustrative plates Chinese traditional medicine, one or more that select following vibrational band as feature with reference to bands of a spectrum:
Raman signatures is with reference to bands of a spectrum
Collection: collect bands of a spectrum wave number, spectral bandwidth, band intensity and bands of a spectrum shape by manual or employing mathematical analysis processing, function fitting method;
Result treatment
Qualitative analysis: contrast test sample, the not guest molecule, the Raman collection of illustrative plates of inclusion compound of inclusion, analyze individual features with reference to bands of a spectrum or feature peak shape, peak width, peak intensity, position with reference to the bands of a spectrum group, basically identical judges whether inclusion;
Key band or the qualitative identification of key band group and the inclusion state of analysis medicine in cyclodextrin system or preparation in the Raman collection of illustrative plates of selection drug molecule, the variation of stripping parameter, solubility parameter, permeability parameters and the pharmacokinetic parameters of qualitative judgement medicine.
According to the interaction of described medicine of claim 1 and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof, it is characterized in that 2, described sample to be measured comprises: liquid, solid, semisolid;
The content range of system Chinese traditional medicine is 0.1%～80%.
According to claim 1 or the interaction of 2 described medicines and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof, it is characterized in that 3, increasing has following steps:
One of following method is adopted in quantitative test,
(1) criterion keying method, be provided with and the approaching standard control system of working sample condition, from described feature with reference to selected characteristic absorption peak the bands of a spectrum, with characteristic peak width, intensity, shift value, bands of a spectrum shape coefficient is index, determine the optimum value of index, choose the ratio of the spectroscopic data of characteristic peak index in sample and the reference substance collection of illustrative plates, carry out all kinds of parameters of calculation sample by following formula respectively; Inclusion degree, stripping parameter, solubility parameter, permeability parameters, pharmacokinetic parameters:
Inclusion degree or inclusion rate=(Г s/ Г r) * 100%,
Г s is the spectral bandwidth of sample with reference to the peak, and Г r is the spectral bandwidth of standard reference material with reference to the peak;
Inclusion degree or inclusion rate=(Rs/Rr) * 100%,
Rs is the bands of a spectrum shape similarity coefficient of sample with reference to the peak, and Rr is the bands of a spectrum similarity coefficient of standard reference material with reference to the peak, obtains by the corresponding function of match;
Inclusion degree or inclusion rate=(Δ fs/ Δ fr) * 100%,
Δ fs is the shift value of sample with reference to the peak, and Δ fr is the shift value of standard reference material with reference to the peak;
Inclusion degree or inclusion rate=[(1-Is)/(1-Ir)] * 100%,
Is is the peak strong coefficient of sample with reference to the peak, and Ir is the peak strong coefficient of standard reference material with reference to the peak;
Medicine stripping parameter PDs (t)=inclusion degree or inclusion rate * PDr (t),
PDs (t) is the stripping parameter of sample t time, and PDr (t) is the stripping parameter of standard reference material t time;
Medicine permeability parameters Papp/s=inclusion degree or inclusion rate * Papp/r,
Papp/s is the permeability parameters of sample, and Papp/r is the standard reference material permeability parameters;
The r of bioavilability parameter (AUC) s=inclusion degree or inclusion rate * (AUC),
(AUC) s is the bioavilability parameter of sample, and (AUC) r is a standard reference material bioavilability parameter;
The r of blood peak concentration of drug (Cmax) s=inclusion degree or inclusion rate * (Cmax),
(Cmax) s is the blood peak concentration of drug of sample, and (Cmax) r is the blood peak concentration of drug of standard reference material;
The r of peak time (Tmax) s=inclusion degree or inclusion rate * (Tmax),
(Tmax) s is the peak time of sample, and (Tmax) r is the peak time of standard reference material;
(2) working curve method or calibration curve method: the series standard reference substance of choosing different inclusion degree, measure Raman spectrum respectively, make fixed reference feature bands of a spectrum spectrum parameter: width, intensity, shift value, shape similarity coefficient, respectively with respect to the working curve or the typical curve of stripping parameter, solubility parameter, permeability parameters, pharmacokinetic parameters, by the spectrum parameter of mensuration or calculation sample fixed reference feature bands of a spectrum, obtain inclusion degree, stripping parameter, solubility parameter, permeability parameters, the pharmacokinetic parameters of sample according to working curve or typical curve.
According to the interaction of described medicine of claim 3 and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof, it is characterized in that 4, the concrete operations step is:
Adopt excessive cyclodextrin and adopt the higher preparation method's preparation standard contrast inclusion compound of inclusion efficient;
Measure the original collection of illustrative plates of this standard control inclusion compound, select the feature reference peak, collect width, intensity, shift value, shape similarity coefficient;
Selection changes the most responsive spectrum parameter index for the inclusion degree, is optimum value with the spectrum parameter desired value of standard control thing feature reference peak, the inclusion rate of calculation sample;
The stripping parameter of bioassay standard tester, permeability parameters, pharmacokinetic parameters, the stripping parameter of calculation sample, permeability parameters, pharmacokinetic parameters;
According to optimum value, finally determine relative inclusion degree, stripping parameter, permeability parameters, the pharmacokinetic parameters of sample.
According to the interaction of described medicine of claim 4 and cyclodextrin and the Raman spectrum analysis method of pharmaceutical property thereof, it is characterized in that 5, selected key band is: the C=O stretching vibration comprises carboxyl, ketone group or ester group, wave number cm
-1For: 1400-1900.
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| 应用光谱分析技术研究碘与-环糊精结合物的结构特征. 宋占军等.光谱学与光谱分析，第21卷第5期. 2001 * |
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