CN105717264A - Method for measuring content of effective ingredients in cinobufagin extraction process - Google Patents
Method for measuring content of effective ingredients in cinobufagin extraction process Download PDFInfo
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Abstract
The invention discloses a method for measuring the content of effective ingredients in the cinobufagin extraction process. The method comprises steps as follows: step 1) no fewer than 100 parts of cinobufagin extracting solution samples obtained in the extraction processes of different batches are collected; step 2) key indexes of the cinobufagin extracting solution samples are measured with a conventional analysis method respectively; step 3) a quantitative model between near infrared spectroscopy and each key index is established; step 4) the quantitative model is verified; step 5) key indexes of effective ingredients in an unknown sample are rapidly measured. The provided method for measuring the content of the effective ingredients in the cinobufagin extraction process can effectively solve the problem about rapid detection of the content of the effective ingredients in the cinobufagin extraction process, the cinobufagin extraction end point is rapidly judged, accurate control of process units is improved, finally, the quality stability of cinobufagin extracting solutions in different batches is finally improved, and the final product quality is guaranteed.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, it is more particularly related to a kind of cinobufacin extracts the assay method of process active constituent content.
Background technology
Cinobufacin derives from the skin of Bufo siccus BufobufogargarizansCantor or Bufo melanostictus BufomelanostictusSchneider and obtains through extracting separation; it is that two kind new medicines that China develops voluntarily protect kind with national Chinese medicine; there is the pharmacological action regulating immunity, suppressing tumor cell proliferation, anti-hepatitis virus, the clinical treatment of antitumor, treatment chronic hepatitis B and other diseases is widely used.
The production technology of current cinobufacin is Cutis Bufonis extracting in water, concentration, precipitate with ethanol, secondary concentration, secondary precipitate with ethanol, concentration receipts cream etc., and process of wherein extracting is the key unit operations that cinobufacin produces.Due to composition numerous complicated in Cutis Bufonis extracting solution, carry out quickly measuring relatively difficult to the content of its index Quality Control composition.If lacking effective quality testing means, it is impossible to active constituent content change in monitor in real time extraction process, can cause that production technology hardly results in accurate control, affecting homogeneity and the stability of product.Therefore, research is suitable to the assay method of active constituent content in Cutis Bufonis extraction process, realize, to the quick mensuration of indoles alkaloid, solid content in cinobufacin extraction process, being favorably improved cinobufacin and extracting the quality testing level of production process, it is ensured that the homogeneity of product quality and stability.
Summary of the invention
It is an object of the invention to provide a kind of cinobufacin and extract the assay method of process active constituent content, the method can effectively solve the difficult point of the quickly detection of effective content in cinobufacin extraction process, realize cinobufacin and extract the quick judgement of terminal, to improve the accurate control of process unit, final improve cinobufacin extracting solution quality batch between stability, it is ensured that end product quality.
In order to realize these purposes and the further advantage of the present invention, it is provided that a kind of cinobufacin extracts the assay method of process active constituent content, comprises the steps:
1, a kind of cinobufacin extracts the assay method of process active constituent content, it is characterised in that comprise the steps:
Step 1) collect the cinobufacin extracting solution sample no less than 100 parts obtained in different batches extraction process, described cinobufacin extracting solution sample includes checking collection sample and calibration set sample；
Step 2) adopt traditional analysis to measure the key index of cinobufacin extracting solution sample respectively, including indoles alkaloid content and solid content；
Step 3) screen preprocessing procedures, select suitable spectral band and determine the best main cause philosophical works, the near infrared light spectrogram of acquisition correction collection sample；Set up the quantitative model between near infrared spectrum and each key index；
Step 4) adopt the condition identical with calibration set sample to measure the near infrared light spectrogram of checking collection sample, input built quantitative model, evaluate stability and the predictive ability of the quantitative model set up；
Step 5) adopt the condition identical with calibration set sample to measure the near infrared spectrum data of unknown sample, input built quantitative model and obtain the information of forecasting of each key index.
Preferably, described step 1) in the preparation process of cinobufacin extracting solution sample be: taking clean dry Cutis Bufonis and extract at twice, first time extracts the pure water adding 5 times of Cutis Bufonis, and heating decocts 45min to insulation after boiling；Second time is extracted, and takes first time medicinal residues and adds the pure water of 6 times of Cutis Bufonis, heats to boiling insulation and decocts 30min, wherein, decocts for the first time and collects an extracting solution the pure water of supplementary same volume every 2.5min；Second time decocts collects an extracting solution every 5min, and the pure water of supplementary same volume, repeat n different batches cinobufacin and extract experiment, and obtain no less than 100 parts cinobufacin extracting solution samples, taking wherein a collection of as checking collection sample, itself and n-1 criticize as calibration set sample.
Preferably, described step 2) in the traditional analysis that adopts include Ultraviolet spectrophotometry, oven drying method and weight method, be respectively used to measure indoles alkaloid content and solid content in extracting solution sample.
Preferably, described step 3) near infrared light spectrogram adopt transmission beam method collection, wavelength band is 4000～10000cm-1, scanning times is 32 times, and resolution is 8cm-1, optical fiber transmission-type probe light path is 2mm, and with temperature 25 DEG C, the air of humidity 40% is reference.
Preferably, described step 3) described in preprocess method include: first differential method, second-order differential method, Savitsky-Golay smoothing techniques and Norris derivative filtering method.
Preferably, described step 3) described in quantitative model adopt the partial least square method in stoichiometry software to set up each key index near-infrared quantitative model in extracting solution medicine.
Preferably, described step 4) in judge that the stability of quantitative model set up and the standard of predictive ability are: prediction deviation value is less than 10%.
The present invention at least includes following beneficial effect:
(1) present invention is by pre-building the near-infrared of effective ingredient in cinobufacin extracting solution and the quantitative model of effective ingredient, and it is used in extraction process by this model the quantitative analysis of effective ingredient, realize the quick analysis to whole extraction process, solve the difficult point of the quickly detection of effective content in cinobufacin extraction process, realize cinobufacin and extract the quick judgement of terminal, to improve the accurate control of process unit, final improve cinobufacin extracting solution quality batch between stability, it is ensured that end product quality；
(2) near infrared technology is quickly measured active constituent content in cinobufacin extraction process by the present invention, simple, quick, accurate.Compared with traditional analysis method, minute shortens, and sample need not be carried out pretreatment, saves manpower and materials.
Part is embodied by the further advantage of the present invention, target and feature by description below, and part is also by by being understood by those skilled in the art the research of the present invention and practice.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention will be further described, but the present invention is not limited to this.
Embodiment 1
1, the collection of cinobufacin extracting solution sample
Dry maxima skin 500g, cleans, and extracts at twice: first time extracts, and adds the pure water of 2500ml, heats to boiling insulation and decocts 45min；Second time is extracted, and takes the pure water that first time medicinal residues add 3000ml, heats to boiling insulation and decocts 30min.First time decocts collects an extracting solution 5ml every 2.5min, and mends 5ml pure water.Second time decocts collects an extracting solution 5ml every 5min, and mends 5ml pure water.Repeat 10 different batches cinobufacins and extract experiment, and obtain 240 samples.Randomly selecting wherein batch of data to collect as checking, all the other 9 batch samples participate in modeling as calibration set.
2, the mensuration of cinobufacin extracting solution sample key index
1) mensuration of solid content
To drying the weighing botle (W to constant weight (weight difference is less than 1mg after twice drying)0) in accurately add 2ml extracting solution sample, weigh (W1), water bath method, dry to constant weight (after twice drying, weight difference is less than 1mg) for 105 DEG C, weigh after cooling (W to exsiccator2)；Solid content (%)=(W2-W0)/(W1-W0)。
2) indoles alkaloid (in 5-hydroxy tryptamine hydrochlorate) assay
Precision weighs 5-hydroxy tryptamine hydrochlorate reference substance 15.03mg, puts in 100ml measuring bottle, by dissolved in purified water and be diluted to scale, shakes up, and precision measures 10ml, is placed in 50ml measuring bottle, adds pure water to scale, shakes up, obtains reference substance solution.
Precision measures reference substance solution 1.0,2.0,3.0,4.0,5.0ml, put in 10ml measuring bottle respectively, adding 15% paradime thylaminobenzaldehyde hydrochloric acid (2 → 3) solution 5ml to shake up, add pure water and shake up to scale, room temperature places 35min, according to spectrophotography (" Chinese Pharmacopoeia " 2010 editions, annex VB), with purified water for blank, absorbance is measured at 555nm wavelength place, with absorbance for vertical coordinate, corresponding concentration is abscissa, drawing standard curve chart.The regression equation of curve obtained is Y=0.0389X+0.0276, R=0.9994, it was shown that indoles alkaloid is at 0.139～0.608 μ g ml-1Concentration range internal linear relation is good.
It is appropriate that precision measures cinobufacin extracting solution, centrifugal, it is diluted to suitable multiple, then by the method for regulation under " preparation of standard curve " item, from " adding 15% paradime thylaminobenzaldehyde hydrochloric acid (2 → 3) solution ", measure trap in accordance with the law, standard curve regression equation calculation draw the content of need testing solution indoles alkaloid.
3, the collection of the NIR transmittance spectroscopy of calibration set sample
Using Fourier Transform Near Infrared instrument to gather the near infrared light spectrogram of cinobufacin extracting solution sample, wavelength band is 4000～10000cm-1, and scanning times is 32, resolution is 8cm-1, optical fiber transmission-type probe light path 2mm, with air for reference (temperature 25 DEG C, humidity 40%).
4, the foundation of quantitative model
The partial least square method (PSO-LS-SVM) in stoichiometry software is used to set up the Near-Infrared Quantitative Analysis model of extracting solution sample indoles alkaloid, solid content.Before model is set up, first to calibration set spectrum being carried out abnormity point differentiation to improve model accuracy, spectrum is smoothed, the suitable Pretreated spectra such as differential to be to eliminate instrumental background or the drift impact on signal simultaneously, optimum wave band is selected to extract effective information, determine the suitableeest main gene, by staying a cross verification to judge optimal models parameter.
1) near infrared spectrum preprocess method screening
Original spectrum is carried out respectively that first differential, second-order differential, Savitsky-Golay be smooth and Norris derivative filtering etc., the comparative study various preprocessing procedures impacts on gained model.
2) best main cause subnumber is determined
Best main cause subnumber for quantitative analysis is extremely important to guarantee quantitative model prediction stability, when because causing when subnumber is chosen too small that model information amount is inadequate, because easily causing the Expired Drugs of model when subnumber is chosen excessive.
5, the checking of quantitative model
Taking 24 parts of extracting solution samples of known key index value as checking collection, concrete data are in Table 1；The near infrared spectrum of transmission beam method collection checking collection sample is adopted by calibration set identical parameters, it is input in the near-infrared forecast model set up, between extracting solution near infrared spectrum and each key index, dependency is good, content and the solid content of the indoles alkaloid being verified in collection sample is calculated through model, evaluate stability and the predictive ability of set up quantitative model, it was predicted that deviation value is less than 10%.
6, the quick detection of indoles alkaloid content, solid content in unknown sample
Repeat cinobufacin extract process and obtain new unknown extracting solution sample, according to the near infrared spectrum that step 3 in the present embodiment obtains, spectroscopic data is inputted respectively the quantitative model set up in step 4 in the present embodiment, calculates content and the solid content of the indoles alkaloid being verified in collection sample through model.
Indoles alkaloid content and solid content in table 1 checking collection sample segment
Although embodiment of the present invention are disclosed as above, but listed utilization that it is not restricted in description and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, it is easily achieved other amendment, therefore, under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited to specific details.
Claims (7)
1. the assay method of a cinobufacin extraction process active constituent content, it is characterised in that comprise the steps:
Step 1) collect the cinobufacin extracting solution sample no less than 100 parts obtained in different batches extraction process, described cinobufacin extracting solution sample includes checking collection sample and calibration set sample；
Step 2) adopt traditional analysis to measure the key index of cinobufacin extracting solution sample respectively, including indoles alkaloid content and solid content；
Step 3) screen preprocessing procedures, select suitable spectral band and determine the best main cause philosophical works, the near infrared light spectrogram of acquisition correction collection sample；Set up the quantitative model between near infrared spectrum and each key index；
Step 4) adopt the condition identical with calibration set sample to measure the near infrared light spectrogram of checking collection sample, input built quantitative model, evaluate stability and the predictive ability of the quantitative model set up；
Step 5) adopt the condition identical with calibration set sample to measure the near infrared spectrum data of unknown sample, input built quantitative model and obtain the information of forecasting of each key index.
2. cinobufacin as claimed in claim 1 extracts the assay method of process active constituent content, it is characterized in that, described step 1) in the preparation process of cinobufacin extracting solution sample be: take clean dry Cutis Bufonis and extract at twice, first time extracts the pure water adding 5 times of Cutis Bufonis, and after heating extremely boiling, insulation decocts 45min；Second time is extracted, and takes first time medicinal residues and adds the pure water of 6 times of Cutis Bufonis, heats to boiling insulation and decocts 30min, wherein, decocts for the first time and collects an extracting solution the pure water of supplementary same volume every 2.5min；Second time decocts collects an extracting solution every 5min, and the pure water of supplementary same volume, repeat n different batches cinobufacin and extract experiment, and obtain no less than 100 parts cinobufacin extracting solution samples, taking wherein a collection of as checking collection sample, itself and n-1 criticize as calibration set sample.
3. cinobufacin as claimed in claim 1 extracts the assay method of process active constituent content, it is characterized in that, described step 2) in the traditional analysis that adopts include Ultraviolet spectrophotometry, oven drying method and weight method, be respectively used to measure indoles alkaloid content and solid content in extracting solution sample.
4. cinobufacin as claimed in claim 1 extracts the assay method of process active constituent content, it is characterised in that described step 3) near infrared light spectrogram adopt transmission beam method collection, wavelength band is 4000～10000cm-1, scanning times is 32 times, and resolution is 8cm-1, optical fiber transmission-type probe light path is 2mm, and with temperature 25 DEG C, the air of humidity 40% is reference.
5. cinobufacin as claimed in claim 1 extracts the assay method of process active constituent content, it is characterized in that, described step 3) described in preprocess method include: first differential method, second-order differential method, Savitsky-Golay smoothing techniques and Norris derivative filtering method.
6. cinobufacin as claimed in claim 1 extracts the assay method of process active constituent content, it is characterized in that, described step 3) described in quantitative model adopt the partial least square method in stoichiometry software to set up each key index near-infrared quantitative model in extracting solution medicine.
7. cinobufacin as claimed in claim 1 extracts the assay method of process active constituent content, it is characterised in that described step 4) in judge that the stability of quantitative model set up and the standard of predictive ability are: prediction deviation value is less than 10%.
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