CN113049702B - Quality detection method of radix Puerariae decoction based on fingerprint and its production method - Google Patents
Quality detection method of radix Puerariae decoction based on fingerprint and its production method Download PDFInfo
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Abstract
The invention provides a method for producing kudzuvine root soup based on simplified fingerprint spectrum establishment, which comprises the steps of searching a fingerprint spectrum system with optimal spectrum parameters by using a sample in advance, testing a standard reference sample of a single medicine by using the spectrum system, and searching and determining fingerprint peaks of a small amount of specific components at one time, thereby establishing a simplified qualitative and quantitative fingerprint spectrum model. The simplified fingerprint pattern model can preliminarily qualitatively screen the kudzu root soup products with unqualified quality, and can carry out quantitative analysis on qualified products. Then, the qualified product is comprehensively analyzed by combining a more comprehensive thin-layer chromatography identification method. The invention has the technical effects of simplifying the quality detection process, retaining the effect of accurate quantitative analysis and improving the detection efficiency.
Description
Technical Field
The invention relates to a quality control method of a preparation process of kudzuvine root decoction, in particular to a preparation process of a lily and rehmannia root decoction solid preparation.
Background
Kudzuvine root decoction is originated from Shang Han Lun of Zhang Zhongjing of Han dynasty, is a pungent and warm exterior-releasing agent, and has the effects of inducing sweat to release exterior, promoting body fluid production and relaxing muscles and tendons. The kudzu root decoction particles have the current standard of being collected in the first edition of the 2015 edition of Chinese pharmacopoeia and are used for treating wind-cold type common cold with the symptoms of: fever, aversion to cold, nasal obstruction, nasal discharge, cough, throat itching, thin and white sputum, no sweat, headache, body pain, stiff and uncomfortable neck and back, thin and white or thin and white moist tongue coating, and floating or superficial and tense pulse.
The radix puerariae decoction is prepared from a traditional Chinese medicine classic formula, and comprises 60g of radix puerariae, 45g of ephedra, 30g of cassia twig, 45g of ginger, 30g of honey-fried licorice root, 30g of radix paeoniae rubra and 45g of Chinese date or uniformly mixed according to the weight proportion, wherein 7 of the above materials are added with 2000ml of water or added according to the volume proportion, the radix puerariae and the ephedra are boiled firstly, boiled with strong fire, boiled with slow fire to 1600ml or boiled to the volume proportion, white foam is removed, the rest of the medicinal materials are added, and boiled with slow fire to 600ml or boiled to the volume proportion, so that granules are obtained.
In order to effectively monitor the quality of the traditional Chinese medicine components, the identification and effective content determination of the components of the raw materials are required. The thin layer identification method is a common method in the establishment of a medicine quality standard, during identification, a proper stationary phase is coated on a glass plate, plastic or an aluminum sheet so as to form a uniform thin layer, and after the thin layer is unfolded, the thin layer is transplanted according to a certain ratio and compared with a proper reference substance so as to complete the identification and the content determination of the medicine.
In recent years, the method for detecting the traditional Chinese medicine components by utilizing the fingerprint spectrum technology becomes a high-efficiency and convenient quality detection or monitoring method. The fingerprint technology is to perform liquid mass spectrometry on a traditional Chinese medicine extraction solution by using High Performance Liquid Chromatography (HPLC), and form a fingerprint specific to the traditional Chinese medicine through specific peak lines and combinations of the peak lines of the chromatogram characterizing specific components of the traditional Chinese medicine, thereby completing quality detection or monitoring of the traditional Chinese medicine preparation.
For example, shihong congratulation (HPLC fingerprint and multi-index quantitative analysis of pueraria decoction, journal of chinese experimental and prescriptions, 2015, 15 th year) discloses establishing a fingerprint of pueraria decoction, analyzing 38 common peaks, comparing the peak positions of a reference substance, a single medicinal material, a negative sample and a pueraria decoction sample, obtaining attribution from 35 common peaks, obtaining chemical component identification from 10 common peaks, and selecting 14 index components closely related to the activity of the reference substance against primary dysmenorrhea. Although the literature comprehensively analyzes all identifiable fingerprint peaks of the pueraria decoction, the identification indexes are too many, the data processing amount required to be analyzed and corrected is too large, and the method is not suitable for quickly and primarily identifying the fingerprint of the pueraria decoction.
The prior patent application CN201711009683.7 of the applicant and the inventor, and the preparation process of the Chinese medicinal preparation disclose that the fingerprint is used for identifying and analyzing various preparations including the kudzuvine root decoction. The method comprises selecting characteristic fingerprint spectra of the prepared XIAOCHAIHU decoction granule extractive solution according to HPLC mass spectrum peaks of bupleuri radix, Scutellariae radix, Ginseng radix, rhizoma Pinelliae Preparada, radix Glycyrrhizae Preparata, rhizoma Zingiberis recens, and fructus Jujubae, so as to establish respective characteristic fingerprint spectra for 7 components respectively, and obtain good detection effect. Based on the detection method, the invention also establishes a new preparation method of the small bupleurum decoction preparation. However, the invention essentially discloses the establishment process of the fingerprint model of the bupleurum tenue decoction, and does not disclose the fingerprint model of the pueraria tenuifolia decoction, and simultaneously needs to test respective fingerprint characteristic peaks of 7 medicinal flavors through a large number of tests, so that the characteristic fingerprint peak with identification significance and the application thereof are not disclosed, which brings complexity and inconvenience to the establishment process of the fingerprint. Therefore, the method also does not meet the requirement of rapid preliminary qualitative identification by using the fingerprint of the kudzuvine root decoction in the prior art.
As for the fingerprint spectrum research of other traditional Chinese medicine preparations, for example, zhengru (high performance liquid phase fingerprint spectrum research of bupleurum tenue granules, evaluation and analysis of the medication in chinese hospital, vol.17, No. 8, 2017) reported that the fingerprint spectrum of bupleurum tenue granules and 7 traditional Chinese medicines (codonopsis pilosula, liquorice, jujube, ginger, bupleurum, scutellaria baicalensis, pinellia ternate) composed of the same was comparatively analyzed by HPLC, and the distribution of 26 peaks among 35 common peaks in the common pattern of the fingerprint spectrum of the bupleurum tenue granules in the 7 traditional Chinese medicines was studied; according to the common peak characteristics corresponding to each medicinal material, the using condition of the bupleurum tenue particle Chinese medicinal material can be reflected to a certain extent, and the product quality can be distinguished from the medicinal material aspect. However, this method needs to test the common characteristic peaks through a large number of tests, and the number of detected fingerprint peaks is as high as 26, which causes complexity and inconvenience to the process of establishing the fingerprint.
Weihuizhen (Maxingshigan decoction multi-wavelength switching fingerprint spectrum research, Vol.23, No. 1 of Shizhen national medicine, 2012) reports that Agilent1100-DAD detector is utilized, the mobile phase is acetonitrile-0.1% phosphoric acid, gradient elution is carried out, the detection wavelength is 0-32min:215 nm; 32-40min, 350 nm; 254nm for 40-50 min; the sample volume is 10 mul, the column temperature is 25 ℃, the flow rate is 1ml/min, 12 common fingerprint peaks in the Maxingshigan decoction are determined, and an HPLC fingerprint pattern model is established. The method can reflect the use condition of the traditional Chinese medicine of the Maxingshigan decoction to a certain extent, and can distinguish the product quality from the perspective of the medicinal materials. However, this method requires a large number of tests to test the common characteristic peaks, and at the same time, the number of detected fingerprint peaks is as large as 12, which causes a cumbersome and inconvenient process for creating a fingerprint.
The invention relates to a Chinese granted invention patent CN201510597933.8, the invention name of which is a detection method of ephedra, gypsum and licorice decoction formula particles, and discloses a method for detecting the quality of the ephedra, gypsum and licorice decoction by infrared fingerprint, thin-layer qualitative identification and HPLC content measurement, wherein ephedrine hydrochloride, pseudoephedrine hydrochloride and amygdalin are taken as characteristic peaks to establish an HPLC fingerprint model to obtain better detection effect. However, the method needs to perform preliminary screening on infrared fingerprint and thin-layer layered analysis in advance to obtain fewer 3 characteristic fingerprint peaks, and the whole process is also complicated and inconvenient for establishing the fingerprint.
Therefore, in the prior art, for completing quality detection or monitoring of traditional Chinese medicines by using a fingerprint spectrum and a traditional Chinese medicine preparation method established by using the method, characteristic fingerprint peaks of a plurality of medicinal flavors are required to be found and established generally. However, as many as 7 kinds of ingredients can be contained in the pueraria decoction, and as many as hundreds of kinds of active ingredients can be detected. Considerable screening work is required if the different active ingredients in the finished granules are identified one by one and the characteristic fingerprint peaks are determined. Therefore, more technical obstacles are brought to establishing the fingerprint detection method of the kudzuvine root decoction.
Therefore, there is a need for a method for quality monitoring of pueraria decoction product by fingerprint spectrum method, and a need for a standardized production method based on the quality monitoring method.
Disclosure of Invention
The principle of the invention is as follows: the invention breaks through the traditional research mode that a fingerprint model enough for representing various specific medicinal materials can be established only by repeatedly searching and screening suitable fingerprint peaks for multiple components in the traditional Chinese medicinal composition, and firstly proposes: the fingerprint spectrum system with the best spectrum parameter is searched by using a standard sample in advance, then the spectrum system is used for testing a standard control sample of a single medicine, and fingerprint peaks of a few specific components are searched and determined at one time, so that a simplified qualitative and quantitative fingerprint spectrum model is established. The simplified fingerprint pattern model can preliminarily qualitatively screen the kudzu root soup products with unqualified quality, and can carry out quantitative analysis on qualified products. And then, the qualified quality product is comprehensively analyzed by combining a more comprehensive thin-layer chromatography identification method, so that the technical effects of simplifying the quality detection process, retaining the effect of accurate quantitative analysis and improving the detection efficiency are achieved.
Therefore, the first objective of the present invention is to provide a method for rapidly detecting pueraria decoction by combining fingerprint and thin layer chromatography, comprising the steps of:
(1) establishing a simplified qualitative fingerprint spectrum mass spectrum model, which comprises the following steps:
weighing a proper amount of 10 batches of kudzu root soup particles, adding 50ml of water to dissolve the samples and fixing the volume to obtain a solution of a product to be detected
Secondly, sample introduction and analysis are carried out on each solution to be measured;
analyzing fingerprint parameters of the solution to be detected, selecting chromatographic conditions that the puerarin fingerprint peak appears in about 20min and other peaks appear in about 24min, and establishing a simplified standard fingerprint model;
(2) taking about 0.2g of sample particles, precisely weighing, placing in a 50ml volumetric flask, adding water for dissolving, fixing the volume, shaking up, filtering, and taking a subsequent filtrate;
(3) carrying out sample introduction detection on the test solution to obtain a chromatogram;
(4) comparing whether effluent chromatographic peaks and mAU values of the sample solution and the qualitative fingerprint model in about 20min and about 24min of peak time are consistent, wherein mAU is an abbreviation of milliabsorbance Unit (mAbsorbent Unit), if so, carrying out next detection, and if not, directly judging that the sample to be detected is unqualified;
(5) taking a sample to be detected which passes through the quality primary screening, adding an extracting agent into a filtrate of the sample to be detected for extraction, and combining extract liquor;
(6) evaporating the extractive solution in water bath, and dissolving the residue with methanol to obtain sample solution; preparing positive control solution and negative control solution in parallel by the same method;
(7) respectively sucking a test solution, a positive control solution and a negative control solution, respectively placing the test solution, the positive control solution and the negative control solution on the same silica gel G thin-layer plate, respectively developing the test solution, the positive control solution and the negative control solution on a thin-layer identification developing agent, and taking out the test solution;
(8) adding color developing solution, heating with hot air until the color development of spots is clear, and inspecting in sunlight or ultraviolet light;
(9) comparing the spots of the test sample, the positive control and the negative control, and if the spots of the same color appear at the corresponding positions of the test sample and the positive control and the spots of no interference appear at the negative position, judging that the test sample contains the medicine components with the same or similar quality as the positive control;
wherein the content of the first and second substances,
for radix Puerariae, herba Ephedrae, Glycyrrhrizae radix, and radix Paeoniae Rubra, chloroform-methanol-water (30:10:0.8) is used as developing agent;
for ramulus Cinnamomi and rhizoma Zingiberis recens, petroleum ether (60-90 deg.C) -chloroform-ethyl acetate (2:1:1) is used as developing agent;
in one embodiment, the step two is to carry out sample injection detection according to the following parameters,
the instrument comprises: agilent1100, column: an Agela Venusil MP C18 column (4.6X 250mm, 5 μm);
column temperature: 30 ℃; flow rate: 1 ml/min; detection wavelength: 250 nm;
mobile phase: acetonitrile-water;
further, the process of determining consistency in step (4) includes: taking the corresponding peak of the puerarin reference substance peak as an S peak, and calculating the relative retention time and the relative peak area of each main peak and the S peak; wherein the similarity between the test sample fingerprint and the comparison fingerprint is not less than 0.90, and the relative retention time and the relative peak area are within +/-10% of the specified value, which indicates that the sample to be detected meets the primary quality standard, and if the standard is not met, the sample to be detected is directly judged to be unqualified.
Further, in the step (6), adding a proper amount of water into the kudzuvine root, the ephedra herb, the liquorice, the red paeony root, the cassia twig and the ginger respectively, decocting for 1 hour, filtering, concentrating the filtrate to 20ml, placing the filtrate in a separating funnel, extracting with ethyl acetate for 2 times, 15ml each time, collecting an ethyl acetate layer, evaporating in a water bath, and adding 1ml of methanol into residues for dissolving;
further, in the step (7), respectively sucking 2 μ l of each of the test solution and the control solutions of herba Ephedrae, Glycyrrhrizae radix, and radix Paeoniae Rubra, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (30:10:0.8) as developing agent, taking out, air drying, and analyzing brilliant blue fluorescent spot at 365nm wavelength; or spraying vanillin sulfuric acid solution, heating at 105 deg.C until the color of spots is clear, and analyzing the spots in the spectrum.
Further, in the step (7), respectively sucking 2 μ l of each of 6-gingerol reference substance solution, cassia twig reference medicinal material solution and test sample solution, respectively dropping on the same silica gel G thin layer plate, developing with petroleum ether (60-90 ℃) -chloroform-ethyl acetate (2:1:1) as developing agent, taking out, air drying, and analyzing bright blue fluorescent spots at 365nm wavelength; or spraying vanillin sulfuric acid solution, heating at 105 deg.C until the color of spots is clear, and analyzing the spots in the spectrum.
The second purpose of the invention is to provide a preparation method of a kudzuvine root decoction preparation, which comprises the steps of adding water into 7 medicines for decoction, filtering, merging decoction, decompressing and concentrating filtrate into clear paste, adding a proper amount of maltodextrin, drying or crushing to prepare kudzuvine root decoction particles, carrying out quality detection on the prepared particles, and guiding the product to be produced in a standardized way according to the quality detection result, wherein the quality monitoring or detection step is characterized by comprising the following steps:
the above steps (1) to (9).
Further, the method comprises the following steps of respectively mixing the components in a weight ratio of 6: 4.5: 3: 4.5: 3: 3: 4.5, weighing decoction pieces of radix puerariae, ephedra, cassia twig, ginger, honey-fried licorice root, red paeony root and Chinese date.
Further, the kudzuvine root and the ephedra are firstly decocted by adding water, then other decoction pieces are added, and the decoction pieces are heated and boiled for 1.5 hours;
further, filtering the slightly boiled extracting solution, and concentrating the slightly boiled extracting solution to a relative density of 1.01-1.20 through reduced pressure low temperature concentration;
further, adding or not adding auxiliary materials into the concentrated solution, uniformly mixing, and quickly drying into powder through spray drying;
further, the obtained powder is prepared into granules by wet granulation, dry granulation or boiling granulation;
further, the chromatographic analysis is carried out on the product according to the fingerprint chromatographic model established in the step 1-4. If the analysis result proves that the puerarin product contains an expected puerarin peak and another characteristic fingerprint peak, the product is judged to be preliminarily qualified, the full component detection of the next procedure is carried out, if the expected fingerprint peak is not found, the product is judged to be unqualified, the production is stopped, and all raw materials and production procedures are checked;
further, according to the determined 2 developing agent combinations in the steps 5-9, carrying out thin layer chromatography identification on the kudzu root, the ephedra herb, the cassia twig, the ginger, the honey-fried licorice root, the red paeony root and the Chinese date, determining whether the product meets the quality standard requirement or not according to the identification result, and carrying out subsequent procedures such as total mixing and the like if the product is judged to be qualified according to the thin layer chromatography result. If the thin layer chromatography result is judged to be unqualified, the production is stopped, and all raw materials and production procedures are checked.
Further, the granules are subpackaged into 5 g/bag and packaged by a composite film.
Further, the quality monitoring includes detecting and evaluating the quality of the product, judging whether the product quality is qualified or not, thereby determining whether to continue or interrupt the production process of the product, and guiding the product to be produced in a standardized manner.
Compared with the traditional process, the invention has the following advantages:
1. the existing fingerprint spectrum method needs to determine the characteristic spectrum of various components in the traditional Chinese medicine. Corresponding medicinal materials of the invention can be as many as 7 kinds, and can be used as components of a characteristic spectrum, namely, fingerprint peaks of more than hundreds of kinds, so that great technical difficulty exists in identifying the medicinal materials by fingerprint spectrums. The invention has the creativity that: the fingerprint chromatogram system with the best chromatogram parameters is searched in advance, and then fingerprint peaks of a small amount of specific components are searched and determined at one time through the determined fingerprint chromatogram system, so that a simplified fingerprint model is established. The method has the function of primary screening detection, and the qualified product is subjected to comprehensive thin-layer chromatography, and the unqualified product is directly eliminated. The invention can avoid the tedious establishing process of searching the fingerprint, and avoid the unnecessary waste caused by the accurate quality detection of the comprehensive thin-layer chromatography;
2. in order to further improve the detection stability of the chromatographic primary screening system, a preliminary test for measuring chromatographic conditions and system applicability is introduced before the detection method of the fingerprint quality primary screening is carried out, and the stability and the accuracy of the fingerprint chromatographic model are verified through a large number of tests;
3. in order to further detect whether all the medicines are contained in the composition, on the basis, the primary screening system is further combined with a predetermined more comprehensive TLC detection method to perform qualitative and quantitative analysis on the qualified medicine composition again, so as to determine whether all the medicines meeting the quality requirements are contained. The particle thin-layer chromatography is used for identifying 6 medicinal materials in the prescription by adopting 2 developing systems instead of jujube as an adjuvant, and is more efficient than identifying 4 medicinal materials under 4 developing conditions recorded in Chinese pharmacopoeia;
4. in the quality detection method, a large number of specific detection data point values are involved, although the protection range is narrower, unqualified products with poor or insufficient effect are eliminated through various specific detection data point values, so that the quality detection method is very suitable for establishing stable quality control standards and meets the requirements of traditional Chinese medicine production;
5. the invention comprehensively monitors the product quality by using detection methods such as a fingerprint spectrum mass spectrum model, a thin layer chromatography, improved content measurement and the like, and the detection method is stricter than a pharmacopoeia method so as to ensure marketization of finished medicines.
Drawings
FIG. 1: ultraviolet spectrogram of puerarin reference substance in example 1;
FIG. 2: the test sample spectra prepared from water, methanol and ethanol in example 1;
FIG. 3: sample maps of different extraction methods in example 1;
FIG. 4: the standard fingerprint of the kudzuvine root decoction determined in the embodiment 1;
FIG. 5: the test sample, negative control and puerarin control maps in example 2;
FIG. 6: in example 3, spectra of samples of kudzu root and kudzu root soup at 365nm wavelength;
FIG. 7: the vanillin sulfuric acid test solution color spot spectrum of the ephedra herb, the liquorice, the red paeony root and the kudzuvine root soup sample in the embodiment 3;
FIG. 8A: in example 3, spectra were obtained on samples of cinnamon twig, kudzu and kudzu soup at 365 nm;
FIG. 8B: the vanillin sulfuric acid test solution color spot spectrum of the ginger and kudzuvine root soup sample in the example 3;
FIG. 9: example 5 is a schematic flow chart of the preparation of pueraria decoction involving quality control.
Detailed Description
The following examples are provided to further illustrate the technical solutions of the present invention, but the examples are only for illustrating the present invention and are not to be construed as limiting the present invention.
Example 1 determination of optimization conditions for finger-print chromatography
The quality control of the preparation process of the kudzuvine root decoction particles based on the fingerprint spectrum is carried out by the following steps:
grouping experiment 1.1 chromatographic conditions and system suitability preliminary test:
1.1 chromatographic column selection: because the puerarin to be measured is a flavonoid component, a chromatographic column using octadecylsilane chemically bonded silica as a filler is selected, and the chromatographic column used in the test has the following components: a is Agela Venusil MPC18
(4.6X 250mm, 5 μm); b is Phenomenex Gemini C18
(4.6X 250mm, 5 μm); c is as follows: techway JADE-PAK ODS-AQ (4.6X 250mm, 5 μm).
1.2 detection wavelength selection:
dissolving puerarin reference substance with methanol, performing ultraviolet scanning in the range of 200-400nm, and recording ultraviolet scanning spectrum, with reference to FIG. 1, wherein FIG. 1 is an ultraviolet spectrum of puerarin reference substance;
according to the ultraviolet scanning result, the puerarin reference substance has larger absorption at the wavelengths of 250nm and 305nm, the pharmacopeia method selects 250nm for determination, and then 250nm in the pharmacopeia method is kept as the detection wavelength of the test substance.
1.3 determination of chromatographic conditions: the chromatographic conditions for measuring the puerarin content in the kudzuvine root decoction particles are determined by the improvement of the mobile phase and the comparison of the detection wavelength:
a chromatographic column: c18 column (4.6X 250mm, 5 μm);
column temperature: 30 ℃;
flow rate: 1 ml/min;
detection wavelength: 250 nm;
mobile phase: acetonitrile-water;
grouping experiment 1.2 selection of extraction solvent:
taking about 0.2g of a sample (batch number: 20200220), precisely weighing, placing in a triangular flask with a plug, precisely adding 50ml of water, methanol and ethanol respectively, sealing the plug, weighing, respectively performing ultrasonic treatment (250W, 40KHz) for 30min, cooling, weighing again, complementing weight loss reduction with corresponding solvents, shaking up, filtering with a microporous filter membrane, taking a subsequent filtrate, and measuring the content by liquid chromatography, wherein the results are as follows:
referring to fig. 2, fig. 2 is a graph of a sample prepared from water, methanol and ethanol, and it can be seen from the results in fig. 2 that the water extraction effect is the best, the methanol is the second, and the ethanol extraction effect is not good, so that water is selected as the extraction solvent.
Grouping experiment 1.3 different extraction methods investigate:
taking about 0.2g of a sample (batch number: 20200220), paralleling 2 parts, precisely weighing, placing in a triangular flask with a plug, precisely adding 50ml of water, sealing the plug, weighing, respectively performing ultrasonic treatment (250W, 40KHz), heating reflux, shaking for extraction for 30min, cooling, weighing again, supplementing water to reduce weight loss, shaking up, filtering with a microporous membrane, taking a subsequent filtrate, measuring the content with liquid chromatography:
referring to fig. 3, fig. 3 is a sample map of different extraction methods, and the result shows that 3 extraction methods are used, the sample content is basically consistent, so that the sample can meet the requirement as long as the sample is dissolved, and the preparation method is changed to be adding 50ml of water to dissolve the sample.
1.4 Final optimization parameters of the fingerprint method:
the optimal fingerprint conditions obtained according to the above experiment are as follows, and the standard fingerprint is shown in FIG. 4:
extracting solvent: water;
the instrument comprises: agilent1100
And (3) chromatographic column: an Agela Venusil MP C18 column (4.6X 250mm, 5 μm);
column temperature: 30 ℃;
flow rate: 1 ml/min;
detection wavelength: 250 nm;
mobile phase: acetonitrile-water;
referring to fig. 4, fig. 4 is a fingerprint of pueraria decoction standard, showing that the peak time of the fingerprint of puerarin is about 20min, and the peak time is about 24 min.
Example 2: verifying the determined standard fingerprint model and detecting a sample;
2.1 verification accuracy of standard fingerprint model
19.70mg of puerarin contrast (batch No. 110752-201514, purity 95.5%, China institute for food and drug testing) is precisely weighed, and methanol is added to dissolve and fix the volume to 20ml, thus obtaining the control stock solution I with the concentration of 0.9408 mg/ml. Precisely measuring reference substance stock solutions I0.5 ml, 1.0ml and 1.5ml, placing in a conical flask with a stopper, precisely measuring 0.1g of a test substance with a known content (lot number: 20200220, content 9.6mg/g), placing in the conical flask, measuring the content according to a proposed content measurement method, and calculating the recovery rate of puerarin to be 100.0% and RSD to be 0.80% (n to 9), the results are detailed in table 1:
TABLE 1 sample application and recovery rate test result table for pueraria root decoction particles
As shown in table 1, the detection method of the mass spectrometry model is good in accuracy.
2.2 verification of repeatability and precision for standard fingerprint model
2.2.1 repeatability
The same test sample (batch No. 20200220) was precisely weighed 6 parts, and the content was measured by the content measurement method, and the result showed that the puerarin content was 9.6mg/g and RSD was 0.45% (n was 6), indicating that the method had good reproducibility. The results are shown in Table 2:
TABLE 2 results of the repeatability tests
2.2.2 intermediate precision
The same sample (lot No. 20200220) was sampled and tested by different experimenters in 6 replicates according to the requirements of the repeatability test using different instruments, resulting in a content of 9.63mg/g, RSD 0.45% (n 12), indicating good intermediate precision, the results are shown in table 3:
TABLE 3 results of intermediate precision tests
2.3 qualitative preliminary screening detection of fingerprint pattern model for multiple samples
According to the above method, puerarin control solution, radix Puerariae decoction granule sample solution and Scutellariae radix negative control solution are prepared.
Simultaneously measuring puerarin control solution, radix Puerariae decoction granule test solution and Scutellariae radix negative control solution by content measurement method, and comparing the spectra, wherein the detailed spectra are shown in FIG. 5, and FIG. 5 is the spectra of the test sample, negative control and puerarin control;
wherein, the similarity with the established qualitative fingerprint model is calculated according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A version): taking the corresponding peak of the baicalin reference substance peak as an S peak, and calculating the relative retention time and the relative peak area of the main peak and the S peak of the test substance; wherein the similarity between the test sample fingerprint and the comparison fingerprint is not less than 0.90, and the relative retention time and the relative peak area are within +/-10% of the specified value, which indicates that the sample to be detected meets the primary quality standard, and if the standard is not met, the sample to be detected is directly judged to be unqualified.
The result shows that the chromatogram of the test solution presents the same chromatographic peak at the retention time corresponding to the puerarin chromatographic peak, the similarity exceeds 0.95, the separation effect is good, and the chromatogram of the baical skullcap root negative control solution does not present the same chromatographic peak at the retention time corresponding to the puerarin chromatographic peak, thereby indicating that the negative is not interfered.
Example 3, exploration and screening of different extraction solvents (i.e. extractants):
3.1. instruments and reagents
3.1.1 Instrument
A thin-layer automatic imager (CAMAG TLC visual 2, CAMAG, switzerland), a thin-layer plate heating plate (CAMAG TLC PLATE HEATER iii, CAMAG, switzerland), an electronic balance (METTLER ME2002E, mettler-toledo instruments (shanghai), an electronic balance (METTLER MS 204TS, mettler-toledo instruments (shanghai), an ultrasonic cleaner (KQ-300DE, ultrasonic instruments ltd, kunshan), a medical centrifuge (xiang instruments H1850 hunan instrument laboratory instrument development ltd), a constant temperature water bath (HWS-26), a plane chromatography spotter (SPDY-1A, nanjing mackoku science instruments ltd), a silica G thin-layer plate (Qingdao ocean chemical plant), and a silica G thin-layer plate (germany).
3.1.2 reagents and reagents
Pueraria reference medicinal materials (batch No. 121551-201805), puerarin reference materials (batch No. 110752-201514), cassia twig reference medicinal materials (batch No. 121191-201605), ephedra reference medicinal materials (batch No. 121051-201005), radix paeoniae rubra reference medicinal materials (batch No. 121093-201804), liquorice reference medicinal materials (batch No. 120904-201519) and 6-gingerol reference materials (batch No. 111833-201806) are purchased from China pharmaceutical biological product identification institute, and other reagents are analytically pure.
Three batches of Kudzuvine root decoction granules are provided by Guangdong Zhidao pharmaceutical science and technology Co.
3.2. Preparation of solutions
3.2.1 preparation of test solutions
Taking 1g of the product, adding 20ml of water, dissolving by ultrasonic, placing in a separating funnel, extracting with ethyl acetate for 2 times, 15ml each time, collecting ethyl acetate layer, evaporating to dryness in water bath, and dissolving residue with 1ml of methanol to obtain sample solution.
3.2.2 preparation of reference drug and reference solution
3.2.2.1 preparation of reference drug solution
Respectively taking control medicinal materials of 1g of kudzuvine root, 1g of ephedra herb, 0.5g of liquorice, 0.5g of cassia twig and 0.5g of red paeony root, respectively adding appropriate amount of water, decocting for 1 hour, filtering, concentrating the filtrate to 20ml, placing the filtrate in a separating funnel, respectively extracting with ethyl acetate for 2 times, 15ml each time, collecting an ethyl acetate layer, evaporating in a water bath to dryness, and dissolving residues with 1ml of methanol to obtain a control medicinal material solution.
3.2.2.2 preparation of control solutions
(1) Taking cinnamaldehyde control, adding ethanol to make into 1 μ l solution per 1ml, and making into control solution.
(2) Adding methanol into 6-gingerol to obtain 0.5mg/ml solution, to obtain 6-gingerol control solution.
3.2.3 preparation of negative control solution
Weighing the prescription of lacking radix Puerariae, lacking herba Ephedrae, lacking Glycyrrhrizae radix, lacking ramulus Cinnamomi, lacking radix Paeoniae Rubra and lacking rhizoma Zingiberis recens according to the prescription proportion, decocting according to standard preparation method, collecting decoction equivalent to 1g granule, and preparing Glycyrrhrizae radix, Scutellariae radix, bupleuri radix and Ginseng radix negative control solution according to the above test preparation method.
3.3. Conditions for developing thin layer
(1) Performing thin layer chromatography (general 0502) test, respectively sucking 2 μ l of test solution and herba Ephedrae, Glycyrrhrizae radix, and radix Paeoniae Rubra reference solution, respectively spotting on the same silica gel G thin layer plate, developing with chloroform-methanol-water (30:10:0.8) as developing agent, taking out, air drying, and making bright blue fluorescent spots visible in radix Puerariae and radix Puerariae decoction sample at 365nm wavelength; spraying vanillin sulfuric acid solution, heating at 105 deg.C until the color of spots is clear, and observing corresponding spots on the chromatogram of herba Ephedrae, Glycyrrhrizae radix, radix Paeoniae Rubra and radix Puerariae decoction, please refer to FIG. 7.
(2) Performing thin-layer chromatography (general rule 0502) test, respectively sucking 6-gingerol control solution, ramulus Cinnamomi control solution and sample solution 2 μ l, respectively dropping on the same silica gel G thin-layer plate, spreading with petroleum ether (60-90 deg.C) -chloroform-ethyl acetate (2:1:1) as developing agent, taking out, air drying, and making bright blue fluorescent spot visible in ramulus Cinnamomi and radix Puerariae decoction sample at 365nm wavelength, please see FIG. 8A; spraying vanillin sulfuric acid solution, heating at 105 deg.C until the color of spot is clear, and displaying corresponding spot on 6-gingerol and radix Puerariae decoction sample, see FIG. 8B).
3.4. And (3) verification of methodology:
taking three batches of pilot samples, respectively preparing a kudzu root negative sample, a cassia twig negative sample, a ephedra negative sample, a licorice negative sample, a red peony root negative sample and a ginger negative sample according to a prescription process, respectively preparing kudzu root, cassia twig, ephedra, licorice, red peony root and ginger negative control solutions according to a test solution preparation method, and carrying out sample application and development according to a standard method, wherein the results refer to 6-8, and the strips in the figure 6 are as follows:
1 kudzu root negative control (ethyl acetate extraction)
2 kudzu root medicine (extraction ethyl acetate)
3 Kudzuvine root decoction granule sample 1 (extracted with ethyl acetate)
4 Kudzuvine root decoction granule sample 2 (Ethyl acetate extraction)
5 Kudzuvine root decoction granule sample 3 (Ethyl acetate extraction)
6 Puerarin reference substance (T: 25 deg.C RH: 58%)
Analysis shows that:
all the kudzu root medicinal materials and samples have clear corresponding main bands with the positive control, and the tailing is not obvious.
② the negative control presents a similar trailing with the test sample, but has no clear corresponding main band, which indicates that the negative control has no interference under the developing agent.
FIG. 7 shows the bands as follows:
1: red peony negative control; 2: red peony root; 3: puerariae decoction granule sample 1;
4: puerariae decoction granule sample 2; 5: puerariae decoction granule sample 3; 6: paeoniflorin control;
7: herba Ephedrae negative control; 8: herba Ephedrae; 9: puerariae decoction granule sample 1;
10: puerariae decoction granule sample 2; 11: puerariae decoction granule sample 3; 12: licorice negative control;
13: licorice root medicinal material; 14: puerariae decoction granule sample 1; 15: puerariae decoction granule sample 2;
16: puerariae decoction granule sample 1; 17 Glycyrrhizaside control (T: 25 ℃ RH: 58%)
Analysis shows that:
all the pueraria decoction granule samples contain single main bands of red paeony root, liquorice and ephedra without tailing, and the samples contain the components of the red paeony root, the liquorice and the ephedra besides puerarin in a figure 6;
all negative controls are not interfered under the developing agent.
FIG. 8 shows the bands: 1 cassia twig negative control (ethyl acetate extraction);
2 cassia twig medicinal material (extracted by ethyl acetate);
3 puerariae decoction granule sample 1 (ethyl acetate extraction);
4 puerariae decoction granule sample 2 (ethyl acetate extraction);
5 puerariae decoction granule sample 3 (ethyl acetate extraction);
6 ginger negative control (ethyl acetate extraction);
75-gingerol control;
8 puerariae decoction granule sample 1 (ethyl acetate extraction);
9 puerariae decoction granule sample 2 (ethyl acetate extraction);
10 puerariae decoction granule sample 3 (ethyl acetate extraction);
(T：25℃ RH：58％)；
analysis shows that:
all the kudzu root decoction granule samples contain single cassia twig and gingerol and have no tailing under the color development of 5% vanillin sulfuric acid, which indicates that the samples contain the components of the cassia twig and the gingerol besides the puerarin in figure 6;
all negative controls are not interfered under the developing agent.
1 Cassia twig negative control (ethyl acetate extraction)
2 ramulus Cinnamomi (ethyl acetate extraction)
3 Kudzuvine root decoction granule sample 1 (extracted with ethyl acetate)
4 Kudzuvine root decoction granule sample 2 (Ethyl acetate extraction)
5 Kudzuvine root decoction granule sample 3 (Ethyl acetate extraction)
6 ginger negative control (ethyl acetate extraction)
75-gingerol control substance
8 Kudzuvine root decoction granule sample 1 (Ethyl acetate extraction)
9 Kudzuvine root decoction granule sample 2 (Ethyl acetate extraction)
10 Kudzuvine root decoction granule sample 3 (Ethyl acetate extraction)
(T：25℃ RH：58％)
Example 4, determining the most suitable parameters of the production process of the pueraria decoction:
preparing decoction pieces, wherein the kudzu root GG-YC-1904-01; herba Ephedrae MH-YC-1910-01; cassia twig GZ-YC-1909-06; radix Glycyrrhizae Preparata GC-YP-1909-12; radix Paeoniae Rubra CS-YP-1911-17; chinese date DZ-YC-1909-07; ginger is commercially available.
Grouping example 4.1 Pilot decoction time study
Taking 6kg of kudzuvine root, 4.5kg of ephedra herb, 3kg of cassia twig, 4.5kg of ginger, 3kg of honey-fried licorice root, 3kg of red paeony root and 4.5kg of Chinese date respectively, firstly adding 8 times of water into the kudzuvine root and the ephedra herb, soaking for 30 minutes, heating and boiling, decocting for 15 minutes, then adding other decoction pieces, heating and boiling, decocting for 1.5 hours, sampling at 0.5 hour, 1 hour and 1.5 hours respectively, and detecting respectively, wherein the results are shown in a table 4:
TABLE 4 pilot extraction time review
As shown in Table 4, the effect of decocting the solid matter for 1.5 hours is slightly lower than that of decocting for 1.0 hour, but the puerarin content and the paeoniflorin content are obviously higher than those of the latter, so that the decoction time of the pilot plant is determined to be 1.5 hours.
Grouping example 4.2 pilot centrifuge feed rate review:
cooling the decoction for 1.5 hr to room temperature, centrifuging at 10000rpm/min, observing the centrifugates obtained at feeding speeds of 100L/h, 200L/h and 300L/h, and respectively detecting, wherein the results are shown in Table 5:
TABLE 5 pilot centrifuge feed Rate investigation
As shown in Table 5, the effect of puerarin content of 300L/h in the pilot centrifuge feed rate was slightly lower than 200L/h, but the solid content was significantly higher than the latter, so the pilot centrifuge feed rate was determined to be 300L/h.
Component example 4.3 the centrifugate was concentrated to produce the final product:
at the temperature: 60-85 ℃, vacuum degree of-0.04-0.08 Mpa, spray drying (air inlet temperature 170 +/-5 ℃, air outlet temperature 80-100 ℃, feeding speed 20-22Hz (5.4-9.0kg/h), finally confirming that the optimal feeding speed is 20-22Hz, and the air inlet temperature is mainly used for saving energy consumption under the condition of drying), taking the concentrated solution and the dried powder for detection, wherein the results are shown in table 6:
TABLE 6 detection of pilot plant extracts, centrifugates, concentrates and spray dried powders
As shown in Table 6, the reaction mixture was decocted in a pilot scale at 1.5h, a centrifugal feed rate of 300L/h, and a temperature: under the production conditions of 60-85 ℃, vacuum degree of-0.04-0.08 Mp and spray drying (air inlet temperature of 170 +/-5 ℃, air outlet temperature of 80-100 ℃ and feeding speed of 20-22Hz), the prepared products of each batch have better solid content, puerarin content and paeoniflorin content and stable quality.
Example five, producing kudzu root decoction by using fingerprint spectrum mass spectrum model and thin layer identification method
According to the results of examples 1-4, the steps of the preparation process of the pueraria decoction are determined as follows, and the specific flow is shown in fig. 9:
(1) respectively mixing the following components in a weight ratio of 6: 4.5: 3: 4.5: 3: 3: 4.5, weighing decoction pieces of radix puerariae, ephedra, cassia twig, ginger, honey-fried licorice root, red paeony root and Chinese date;
(2) firstly, adding 8 times of water into the kudzuvine root and the ephedra, soaking for 30 minutes, heating and boiling with strong fire, decocting for 15 minutes to remove bruise, then adding other decoction pieces, heating and boiling, and decocting for 1.5 hours;
(3) removing impurities and macromolecular substances from the slightly boiled extracting solution through high-speed centrifugation to obtain a clear extracting solution, and then concentrating the clear extracting solution to a relative density of 1.01-1.20 through reduced pressure and low temperature concentration for later use;
(4) adding adjuvants or not, mixing, and spray drying to obtain powder;
(5) preparing the obtained powder into granules by wet granulation, dry granulation or boiling granulation;
(6) according to the determined fingerprint mass spectrum model of the example 1-2, the product is subjected to chromatographic analysis, if the analysis result proves that the product contains an expected radix scutellariae peak and another characteristic fingerprint peak, the product is judged to be initially qualified, and the full component detection of the next procedure is carried out. If the expected fingerprint peak is not found, judging that the product is unqualified, stopping production, and checking all raw materials and production processes;
(7) performing thin layer chromatography identification on radix Puerariae, herba Ephedrae, ramulus Cinnamomi, rhizoma Zingiberis recens, radix Glycyrrhizae Preparata, radix Paeoniae Rubra, and fructus Jujubae according to the determined 2 developing agent combinations of embodiments 3-4;
and determining whether the product meets the quality standard of Chinese pharmacopoeia according to the identification result. And if the quality is judged to be qualified according to the thin-layer chromatography result, performing subsequent procedures such as total mixing and the like. And if the thin layer chromatography result is judged to be unqualified, stopping production. Checking all raw materials and production processes;
(8) and (3) performing total mixing on the products passing the quality detection, subpackaging the granules into 10 g/bag, and packaging with a composite film.
In the quality detection method, a large number of specific detection data point values are involved, although the protection range is narrower, unqualified products with poor or insufficient effect are eliminated through various specific detection data point values, so that the quality detection method is very suitable for establishing stable quality control standards and meets the requirements of traditional Chinese medicine production; meanwhile, the product quality is comprehensively monitored by using detection methods such as a fingerprint spectrum mass spectrum model, thin-layer chromatography, improved content measurement and the like, and the detection method is more strict than a pharmacopoeia method so as to ensure marketization of finished medicines.
Claims (5)
1. A quality detection method of radix Puerariae decoction based on fingerprint is characterized by comprising the following steps: the method comprises the following steps:
(1) establishing a simplified qualitative fingerprint spectrum mass spectrum model, which comprises the following steps:
weighing a proper amount of samples of 10 batches of radix puerariae decoction particles, adding 50ml of water to dissolve the samples and fixing the volume to obtain a solution of a product to be detected;
secondly, sample introduction and analysis are carried out on each solution to be measured;
analyzing fingerprint parameters of the solution to be detected, selecting chromatographic conditions that the puerarin fingerprint peak appears in about 20min and other peaks appear in about 24min, and establishing a simplified standard fingerprint model;
(2) taking about 0.2g of sample particles, precisely weighing, placing in a 50ml volumetric flask, adding water for dissolving, fixing the volume, shaking up, filtering, and taking a subsequent filtrate;
(3) carrying out sample introduction detection on the test solution to obtain a chromatogram;
(4) comparing whether the effluent mass spectrum peak and the mAU value of the sample solution and the qualitative fingerprint pattern model are consistent in the peak-out time of about 20min and about 24min, if so, carrying out next detection, and if not, directly judging that the sample to be detected is unqualified;
(5) taking a sample to be detected which passes through the quality primary screening, adding an extracting agent into a filtrate of the sample to be detected for extraction, and combining extract liquor;
(6) evaporating the extractive solution in water bath, and dissolving the residue with methanol to obtain sample solution; preparing positive control solution and negative control solution in parallel by the same method;
(7) respectively sucking a test solution, a positive control solution and a negative control solution, respectively placing the test solution, the positive control solution and the negative control solution on the same silica gel G thin-layer plate, developing the test solution and the negative control solution by using a developing agent, and taking out the test solution and the negative control solution;
(8) adding color developing solution, heating with hot air until the color development of spots is clear, and inspecting in sunlight or ultraviolet light;
(9) comparing the spots of the test sample, the positive control and the negative control, and if the spots of the same color appear at the corresponding positions of the test sample and the positive control and the spots of no interference appear in the negative control, judging that the test sample contains the medicine components with the same or similar quality as the positive control;
wherein the content of the first and second substances,
for radix puerariae, ephedra, liquorice and red peony root, chloroform-methanol-water with the volume ratio of 30:10:0.8 is used as a developing agent;
for cassia twig and ginger, petroleum ether-chloroform-ethyl acetate with the volume ratio of 2:1:1 is used as a developing agent, wherein the petroleum ether is 60-90 ℃;
and (c) a second step of,
secondly, sample injection detection is carried out according to the following parameters:
the instrument comprises the following steps: agilent1100, column: an Agela Venusil MP C18 column, 4.6X 250mm, 5 μm;
column temperature: 30 ℃; flow rate: 1 ml/min; detection wavelength: 250 nm; mobile phase: acetonitrile-water;
time acetonitrile (%) water (%)
0 12 88
20 12 88
21 80 20
24 80 20
25 12 88
32 12 88。
2. The quality detection method of pueraria decoction established based on fingerprint according to claim 1, wherein: the process of judging consistency in the step (4) comprises the following steps: taking the corresponding peak of the puerarin reference substance peak as an S peak, and calculating the relative retention time and the relative peak area of each main peak and the S peak; wherein the similarity between the fingerprint of the test sample and the standard fingerprint is not less than 0.90, and the relative retention time and the relative peak area are within +/-10% of the specified value, which indicates that the sample to be tested meets the primary quality standard, and if the standard is not met, the sample to be tested is directly judged to be unqualified.
3. The quality detection method of pueraria lobata decoction established based on fingerprint spectrum according to claim 1 or 2, wherein the quality detection method comprises the following steps: in the step (6), the kudzu root, the ephedra herb, the liquorice, the red paeony root, the cassia twig and the ginger are respectively added with a proper amount of water, decocted for 1 hour, filtered, the filtrate is concentrated to 20ml, placed in a separating funnel, respectively extracted with ethyl acetate for 2 times, 15ml each time, an ethyl acetate layer is collected, evaporated in a water bath, and the residue is dissolved by adding 1ml of methanol to serve as a positive control.
4. The quality detection method of pueraria decoction established based on fingerprint according to claim 1, wherein: in the step (7), respectively sucking a sample solution and 2 mul of each of a herba ephedrae, liquorice and radix paeoniae rubra control medicinal material solution, respectively dropping the sample solution and the herba ephedrae, liquorice and radix paeoniae rubra control medicinal material solution on the same silica gel G thin-layer plate, developing by using chloroform-methanol-water as a developing agent, taking out, drying in the air, and analyzing bright blue fluorescent spots at a wavelength of 365 nm; or spraying vanillin sulfuric acid solution, heating at 105 deg.C until the color of spots is clear, and analyzing the spots in the spectrum.
5. The quality detection method of pueraria decoction established based on fingerprint according to claim 1, wherein: in the step (7), respectively sucking 6-gingerol control solution, cassia twig control medicinal material solution and test sample solution by 2 mul, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using the petroleum ether-chloroform-ethyl acetate as developing agent, taking out, airing, and analyzing bright blue fluorescent spots at a wavelength of 365 nm; or spraying vanillin sulfuric acid solution, heating at 105 deg.C until the color of spots is clear, and analyzing the spots in the spectrum.
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