CN118209640A - Poria cocos peel formula granule quality detection method - Google Patents
Poria cocos peel formula granule quality detection method Download PDFInfo
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Abstract
The invention provides a quality detection method of poria peel formula particles, which comprises the steps of determining properties, dry extract yield, thin-layer identification, extract, characteristic spectrum and content of pachymic acid A and pachymic acid B of the poria peel formula particles, limiting the content standard of the poria peel formula particles to be the total content range of pachymic acid A and pachymic acid B of 0.2 mg/g-1.4 mg/g, wherein the thin-layer identification adopts thin-layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the content of the pachymic acid A and the pachymic acid B are all measured by adopting a liquid chromatography. The chromatographic condition of the invention is adopted to carry out liquid phase analysis, which can present the characteristic spectrum of 8 characteristic peaks with better separation degree and clearer, can better carry out the content measurement of the pachymic acid A and the pachymic acid B, further can better distinguish the pachyma particles from the pachyma particles, prevents the pachyma from being used as the pachyma, and can better control the quality of the pachyma formula particles through systemization.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality detection method of poria cocos peel formula particles.
Background
Modern medicines are required to have three characteristics of stability, uniformity, safety and effectiveness, and for Chinese patent medicines, various means are required to be adopted for detection, so that the reliability and stability of detection results are ensured. The formula particles have the advantages of easy carrying, convenient taking and reliable curative effect, and the audience has more and more choices. The formula particle is prepared from single medicinal materials through the procedures of water extraction, concentration, drying, granulation and the like, and compared with the traditional medicinal materials, the morphological characteristics of the formula particle are obviously changed, and the authenticity and quality of the product cannot be obtained by naked eye observation. Therefore, it is necessary to establish a quality evaluation system of the traditional Chinese medicine formula particles so as to comprehensively reflect the inherent quality of the formula particles. The Poria peel is the dried skin of the sclerotium of the fungus Poria cocos (Schw.) Wolf of Polyporaceae, and is harvested and excavated more than 7-9 months, and when the Poria cocos slices and the Poria cocos blocks are processed, the skin which is cut off is collected, dried in the shade, and the effects of inducing diuresis and detumescence are achieved, and the edema is mainly used for treating dysuria. At present, a systematic quality detection method is not formed on the poria cocos peel formula particles, the whole internal quality of the poria cocos peel formula particles cannot be reflected by only adopting the existing detection means to detect the poria cocos peel formula particles, the quality control requirements of the traditional Chinese medicine formula particles cannot be met, and the poria cocos peel particles are difficult to distinguish from the poria cocos peel particles in production and clinical application. Therefore, it is necessary to establish a method for detecting the quality of the poria peel formula particle for controlling the quality of medicinal materials.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a poria cocos skin formula particle quality detection method for better distinguishing poria cocos skin formula particles from poria cocos formula particles and better controlling the quality of the poria cocos skin formula particles.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
a method for detecting the quality of Poria cocos peel formula particles comprises the following detection methods,
Determining the properties, dry extract yield, thin layer identification, characteristic patterns, extract and content of pachymic acid A and pachymic acid B of the pachymic skin formula particles, and limiting the content standard of the pachymic skin formula particles to be 0.2 mg/g-1.4 mg/g of total content of pachymic acid A and pachymic acid B, wherein the thin layer identification adopts thin layer chromatography for identification; the characteristic spectrum and the content measurement of the pachymic acid A and the pachymic acid B are all measured by adopting a liquid chromatography; the extract is measured by a hot dipping method;
the determination of the characteristic spectrum by liquid chromatography comprises: analyzing with liquid chromatograph, taking the solution prepared from Poria skin control medicinal material as reference material solution B, taking the solutions prepared from Poria acid A and Poria acid B reference material as reference mixed solution B, taking the solution prepared from Poria skin granule sample as test material solution B, respectively precisely sucking the reference material solution B, reference mixed solution B and test material solution B, respectively injecting into liquid chromatograph, and measuring to obtain the final product; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica is used as a filler, shimadzu Shim-PACK GIST C-AQ (4.6 mm. Times.250 mm,5 μm); mobile phase: acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table a;
Table a gradient elution procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 40→58 | 60→42 |
| 10～25 | 58 | 42 |
| 25～40 | 58→65 | 42→35 |
| 40～100 | 65→95 | 35→5 |
| 100～105 | 95→40 | 5→60 |
Flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: the time period of 0 to 53 minutes is 242nm, and the time period of 53 to 105 minutes is 210nm.
Poria cocos and poria cocos are different in clinical application of traditional Chinese medicine, in order to prevent the poria cocos from being used as poria cocos skin material to produce formula particles, the formula particles are necessary to be controlled in a quality detection method, the invention has the advantage that the index ratio requirement that the ratio of the sum of peak 1 peak area and peak 3 peak area to peak 6 peak area in the poria cocos formula particle chromatograph is larger than 10 is agreed through a large amount of data analysis, and the poria cocos formula particles can be better distinguished, so that the poria cocos is prevented from being counterfeited in production and clinical application.
In one embodiment, the poria peel formula granule is prepared as follows: decocting Poria cortex decoction pieces 15000g in water, filtering, concentrating the filtrate into fluid extract, adding appropriate amount of adjuvant, drying, adding appropriate amount of adjuvant, mixing, granulating, and making into 1000 g.
In one embodiment, the thin layer chromatography comprises the steps of: (1) preparing a test sample solution a: taking 5.0g of poria cocos peel formula particle sample, grinding, adding 50ml of diethyl ether, carrying out ultrasonic treatment for 10 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution a; (2) preparing a control medicinal material solution a: taking 0.5g of poria peel reference medicinal material, grinding, adding 50ml of diethyl ether, carrying out ultrasonic treatment for 10 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a reference medicinal material solution a; (3) performing thin layer chromatography: the thin layer chromatography conditions were as follows: silica gel H thin layer plate; sample application amount: the solution a of the sample is 10uL, the solution a of the reference medicinal material is 1uL, and the solution a and the reference medicinal material are respectively spotted on the same silica gel H thin layer plate; toluene-ethyl acetate-formic acid (20:5:0.5) solution is taken as a developing agent; color-developing agent: 2% vanillin sulfuric acid solution-ethanol (4:1) mixture was heated at 105℃and inspected in sunlight.
In one embodiment, the hot dip method uses ethanol as a solvent and the range of the extract is determined by a hot dip method under the alcohol-soluble extract determination method.
In one embodiment, the determination of the characteristic spectrum by liquid chromatography further comprises the steps of: (1) preparing a reference solution b of a reference medicinal material: taking 1.0g of poria peel reference medicinal material, adding 10ml of ethanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, and taking a subsequent filtrate as a reference substance solution b of the reference medicinal material; (2) preparing a reference substance mixed solution b: taking a proper amount of reference substances of pachymic acid, pachymic acid A and pachymic acid B, precisely weighing, and adding methanol to prepare reference substance mixed solution B containing 80 mug of pachymic acid, 20 mug of pachymic acid A and 20 mug of pachymic acid B per 1ml respectively; (3) preparing a test sample solution b: taking proper amount of poria peel formula particles, grinding, taking about 3.0g, placing into a conical flask with a plug, adding 10ml of ethanol, sealing, performing ultrasonic treatment for 30 minutes, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution b.
In one embodiment, the liquid chromatography is used for determining poria peel formula particles, 8 characteristic peaks are required to be displayed in the obtained sample chromatogram, the 8 characteristic peaks are required to correspond to 8 characteristic peak retention times in the reference chromatogram of the reference medicinal material, the sample chromatograms are respectively used for representing poria acid B, poria acid A, and poria acid peak 1, and peak 3, and peak 6, respectively correspond to the reference poria acid B, reference poria acid A, and reference poria acid peak retention times, the peak corresponding to the reference poria acid A peak in the sample chromatogram is an S1 peak, the relative retention times of peak 2, peak 4, and S1 peak are calculated, the relative retention times are within + -10% of the specified values, and the specified values are: peak 2 is 0.96, peak 4 is 1.21, peak corresponding to pachymic acid reference peak is S2 peak, and relative retention time of peak 5, peak 7, peak 8 and S2 peak is calculated, wherein the relative retention time is within + -10% of the specified value, the specified value is: peak 5 is 0.91, peak 7 is 1.16, peak 8 is 1.21, and the ratio of the sum of peak 1 and peak 3 peak areas to peak 6 peak area should be required to be greater than 10.
In one embodiment, the determination of the content of pachymic acid A and pachymic acid B by liquid chromatography comprises: performing liquid chromatograph analysis, taking solutions prepared from pachymic acid A and pachymic acid B as reference solutions c, taking solutions prepared from pachymic skin granule samples as test solutions c, precisely sucking the reference solutions c and the test solutions c respectively, respectively injecting into liquid chromatograph, and measuring to obtain the final product; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica as filler (4.6mm. Times.250 mm,5 μm); mobile phase: acetonitrile-0.1% formic acid (59:41) solution is taken as a mobile phase, and gradient elution is carried out according to the specification; column temperature: 35 ℃; sample injection amount: 10. Mu.L; detection wavelength: 252nm.
In one embodiment, the method for determining the content of the pachymic acid A and the pachymic acid B by adopting the liquid chromatography method further comprises the following steps: (1) preparing a reference substance solution: taking a proper amount of a reference substance of the pachymic acid A and the pachymic acid B, precisely weighing, adding methanol to prepare a mixed solution containing 100 mug of the pachymic acid A and 50 mug of the pachymic acid B per 1ml, and taking the mixed solution as a reference substance solution c; (2) preparing a test solution: taking proper amount of poria peel formula particles, grinding, taking about 3.0g, precisely weighing, placing into a conical bottle with a plug, precisely adding 10ml of ethanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing loss weight with ethanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.
Compared with the prior art, the poria peel formula particle quality detection method provided by the application has the beneficial effects that: the quality of the poria peel formula particle is evaluated through research on properties of the poria peel formula particle, dry extract extraction rate, thin-layer identification, extract and characteristic spectrum, and content measurement of the poria acid A and the poria acid B, and through multi-aspect measurement, a solid foundation is laid for stable quality of products, feasible quality standards of the poria peel formula particle can be established, effective control of the quality of the poria peel formula particle is realized, and by adopting chromatographic conditions of the application, a characteristic spectrum with better and clearer separation degree can be obtained, and the method has the advantages of better stability, high precision and better reproducibility. The poria cocos peel decoction pieces are decocted to prepare the poria cocos peel decoction piece formula granule, the average value of the total content of the poria cocos acid A and the poria cocos acid B is 0.661mg/g, and the total content range of the poria cocos acid A and the poria cocos acid B is 0.2 mg/g-1.4 mg/g according to the technical requirements of quality control and standard formulation of traditional Chinese medicine formula granule.
Drawings
FIG. 1 is a TLC chart of sample thin layer chromatography for identifying 3 batches of Poria cocos skin formula particles according to an embodiment of the invention.
FIG. 2 is a diagram showing HPLC characteristics of different extraction methods in investigation of the characteristic spectrum measurement extraction method according to an embodiment of the present invention; s1 is reflux extraction of a characteristic spectrum of a sample solution; s2 is ultrasonic extraction of a characteristic spectrum of the sample solution.
FIG. 3 is a diagram showing HPLC characteristics of different extraction times in the investigation of characteristic spectrum measurement extraction time according to an embodiment of the present invention; wherein S1 is ultrasonic extraction for 90min to obtain a sample solution characteristic spectrum; s2, ultrasonically extracting a characteristic spectrum of the sample solution for 60 minutes; s3, ultrasonic extraction is carried out for 30min on the characteristic spectrum of the sample solution.
FIG. 4 is a graph showing HPLC characteristics of different extraction solvents in a profile determination extraction solvent investigation according to an embodiment of the present invention; s1 is a characteristic spectrum of a sample solution extracted by water; s2 is a characteristic spectrum of a sample solution extracted by ethanol; s3 is a characteristic spectrum of the solution of the test sample extracted by methanol.
FIG. 5 is a graph showing HPLC characteristics of different sample amounts in a sample amount investigation according to an embodiment of the present invention; s1 is a characteristic spectrum of a sample solution to be tested, wherein the sample taking amount of the characteristic spectrum is 3.0 g; s2 is a characteristic spectrum of a sample solution to be tested with a sample taking amount of 2.0 g; s3 is a characteristic spectrum of a sample solution to be tested, the sample taking amount of which is 1.0 g.
FIG. 6 superimposes HPLC feature maps for a specificity test in a feature map determination specificity study of an embodiment of the present invention; wherein S1 is ethanol solvent blank control solution characteristic map, S2 is pachymic acid, pachymic acid A, pachymic acid B control solution characteristic map, S3 is pachyma skin test sample solution characteristic map, and S4 is pachyma skin control medicinal material solution characteristic map.
FIG. 7 is a graph of the overlapping peaks common to the repeatability test in the determination of the characteristic spectrum according to an embodiment of the present invention.
FIG. 8 is a graph of the superposition of peaks common to the precision test in the determination of a characteristic spectrum according to an embodiment of the present invention.
FIG. 9 is a graph of the superposition of peaks common to stability tests in a characteristic spectrum measurement according to an embodiment of the present invention.
FIG. 10 is a graph showing the determination of a superimposed characteristic spectrum of different column temperature surveys (+ -2 ℃) for a characteristic spectrum according to an embodiment of the present invention; wherein S1 is a chromatographic column with a column temperature of 28 ℃, S2 is a chromatographic column with a column temperature of 30 ℃, and S3 is a chromatographic column with a column temperature of 32 ℃.
FIG. 11 is a graph showing the determination of a superimposed characteristic spectrum of different column temperature surveys (+ -5 ℃) for a characteristic spectrum according to an embodiment of the present invention; wherein S1 is a chromatographic column with a column temperature of 25 ℃, S2 is a chromatographic column with a column temperature of 30 ℃, and S3 is a chromatographic column with a column temperature of 35 ℃.
FIG. 12 is a profile determination flow rate test (+ -0.02 ml/min) common peak superposition profile for an embodiment of the present invention; wherein S1 is a chromatographic column with a flow rate of 0.78ml/min, S2 is a chromatographic column with a flow rate of 0.80ml/min, and S3 is a chromatographic column with a flow rate of 0.82 ml/min.
FIG. 13 is a profile determination flow rate test (+ -0.2 ml/min) common peak superposition profile for an embodiment of the present invention; wherein S1 is a chromatographic column with a flow rate of 0.6ml/min, S2 is a chromatographic column with a flow rate of 0.8ml/min, and S3 is a chromatographic column with a flow rate of 1.0 ml/min.
FIG. 14 is a superimposed graph of HPLC characteristics of three batches of Poria cocos skins according to an embodiment of the present invention; wherein, the finished product number of S1 is 220403, the finished product number of S2 is 220402, and the finished product number of S3 is 220401.
Fig. 15 is an overlay of HPLC feature patterns of three batches of poria peel finished products and control medicinal materials, control materials and blank reference materials in an embodiment of the present invention.
FIG. 16 is a graph showing HPLC characteristic spectrum fitting of the poria peel finished product and the reference medicinal material in an embodiment of the invention; wherein the ratio of the sum of the peak 1 and 3 areas to the peak 6 area should be greater than 10.
FIG. 17 is a superimposed HPLC profile of a proprietary test in a content determination proprietary study in accordance with an embodiment of the present invention; wherein S1 is the characteristic spectrum of a poria peel sample, S2 is the characteristic spectrum of a poria acid A and a poria acid B reference substance, and S3 is the characteristic spectrum of a maltodextrin reference substance.
FIG. 18 is a graph showing different concentrations of the pachymic acid A control in a linear range test according to an embodiment of the present invention, wherein the amount of pachymic acid A sample is plotted on the abscissa and the pachymic acid peak area is plotted on the ordinate.
FIG. 19 is a graph showing different concentrations of a comparative product of pachymic acid B in a linear range test according to an embodiment of the present invention, wherein the amount of sample introduced for pachymic acid B is on the abscissa and the peak area of pachymic acid is on the ordinate.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
The embodiment provides a quality detection method of poria peel formula particles, which comprises the following steps of analyzing properties, dry extract yield, thin layer identification, characteristic spectrum, extract and content measurement of pachymic acid A and pachymic acid B of the poria peel formula particles, and limiting the formula particle content standard to be 0.13 mg/g-1.19 mg/g of the total content allowable range of the pachymic acid A and the pachymic acid B, wherein the thin layer identification adopts thin layer chromatography for identification; the characteristic spectrum and the pachymic acid content are all measured by liquid chromatography; the extract was measured by hot dip method.
In this embodiment:
Preparing poria peel formula particles: the standard decoction is prepared from a part of tuckahoe skin traditional Chinese medicine material under the item and medicinal parts according to the technical requirement in the 'Chinese pharmacopoeia' 2020 edition, and comprises the medical plant parts of the family name, the Chinese name, the Latin and the medicinal parts, namely: the product is a formula granule prepared by processing dried outer skin of Wolf sclerotium of Polyporaceae fungus Poria cocos (Schw.) and processing according to main quality index of standard decoction. The specific preparation method comprises the following steps: decocting 15000g of poria cocos peel decoction pieces in water, filtering, concentrating filtrate into clear paste (the paste yield of dry extract is 3% -6%), adding an appropriate amount of auxiliary materials, drying (or drying and crushing), adding an appropriate amount of auxiliary materials, mixing uniformly, granulating and preparing 1000g of the poria cocos peel decoction pieces. The dry extract paste yield is determined as the dry extract paste yield according to the paste yield of 29 batches of standard decoction in the range of 70-130% of the average value. Meanwhile, the paste rate data of investigation, pilot scale test and pilot scale test combined with the preparation process of the product are all in the range of 70% -130% of the average value of the paste rate of the standard decoction. Therefore, the dry extract is assumed to have a paste yield of 3% -6%.
1. Property investigation
Traits: taking three batches of poria peel formula particles, and carrying out mass production on each sample, observing and recording the form, color and smell of the samples, and simultaneously combining the color and smell of the standard soup samples. The product is granule with light grey yellow to yellowish brown color; light smell and slightly bitter taste.
2. Thin layer authentication
Referring to the thin layer identification method in the tuckahoe cortex quantity standard of the traditional Chinese medicine formula granule in Shanghai, tuckahoe skin comparison medicinal materials are used as comparison, the thin layer identification method is established, and through methodology investigation and 25 batches of sample tests, the spots of the test sample are clear, and the negative control sample has no interference, so the method is listed in the tuckahoe formula granule quality standard text [ identification ].
Specifically, the thin layer identification method of the poria peel formula granule comprises the following steps:
sample solution preparation: taking 5.0g of the product, grinding, adding 50ml of diethyl ether, carrying out ultrasonic treatment for 10 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of methanol into the residue to dissolve the residue to obtain a sample solution.
Preparing a control medicinal material solution: preparing about 0.5g of poria peel control medicinal material, and preparing into a control medicinal material solution by the same method.
According to thin layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), 10 μl of sample solution and 1 μl of control medicinal solution are sucked and respectively spotted on the same silica gel H thin layer plate, toluene-ethyl acetate-formic acid (20:5:0.5) solution is used as developing agent, and the developing agent is developed, taken out, dried in the air, sprayed with 2% vanillin sulfuric acid-ethanol (4:1) mixed solution, heated at 105deg.C until the spot color is clear, and inspected under sunlight.
According to the detection method, sample application is carried out on three batches of production sample test solutions, negative sample (maltodextrin) solutions and control medicinal material solutions, and thin-layer identification is carried out. As shown in FIG. 1, the results indicate that the same color of main spots appear in the three batches of mass-produced sample chromatograms at positions corresponding to the control medicinal material chromatograms.
3. Feature map
3.1 Feature map measuring method
3.1.1 Chromatographic conditions and System applicability experiments octadecylsilane chemically bonded silica was used as filler (Shimadzu Shim-PACK GIST C-AQ (4.6 mm. Times.250 mm,5 μm)); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the specifications in Table 1; the flow rate is 0.8ml per minute; the column temperature is 30 ℃; detection wavelength: the time period of 0 to 53 minutes is 242nm, and the time period of 53 to 105 minutes is 210nm. The theoretical plate number is not less than 25000 calculated by pachymic acid A peak.
TABLE 1 gradient elution procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 40→58 | 60→42 |
| 10～25 | 58 | 42 |
| 25～40 | 58→65 | 42→35 |
| 40～100 | 65→95 | 35→5 |
| 100～105 | 95→40 | 5→60 |
3.1.2 Preparation of reference solution
Taking 1.0g of poria peel reference medicinal material, adding 10ml of ethanol, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30 minutes, cooling, filtering, and taking the subsequent filtrate as reference substance solution of the reference medicinal material.
And taking proper amounts of reference substances of pachymic acid, pachymic acid A and pachymic acid B, precisely weighing, and adding methanol to prepare reference substance mixed solutions containing 80 mug of pachymic acid, 20 mug of pachymic acid A and 20 mug of pachymic acid B per 1ml respectively.
3.1.3 Preparation of sample solutions
Grinding the above materials, collecting about 3.0g, placing into conical flask with plug, adding ethanol 10ml, sealing, ultrasonic treating (power 300W, frequency 40 kHz) for 30min, cooling, shaking, filtering, and collecting filtrate.
3.1.4 Assays
Respectively precisely sucking 10 μl of reference solution and sample solution, and injecting into liquid chromatograph for measurement.
3.2 Methodology investigation of feature map determination
The method for pre-treating the sample of the characteristic spectrum of the poria cocos peel formula particle (batch number: 220401) is examined, and the influence of the extraction solvent, the extraction mode, the extraction time and the extraction solvent dosage (or the sampling amount) on the characteristic spectrum of the poria cocos peel formula particle is mainly examined.
3.2.1 Investigation of the extraction method
Test solutions were prepared by different extraction methods, and the measurement was performed according to the method described under "3.1" above. As shown in fig. 2 and table 2, the results show that the sample solutions with different extraction modes have consistent numbers of main chromatographic peaks and insignificant peak shape differences, but the reflux extraction method has better extraction effect than the ultrasonic extraction method, so that the reflux extraction is selected as the sample extraction mode.
TABLE 2 comparison of extraction methods
3.2.2 Extraction time investigation
Test solutions were prepared at different times, and the measurement was performed according to the method described under "3.1" above. As shown in FIG. 3 and Table 3, the results show that the sample solutions with different extraction times have the same number of main chromatographic peaks, small peak shape difference and similar extraction effects, so that the extraction time of the sample is 30 minutes for shortening the detection time.
TABLE 3 comparison of extraction times
3.2.3 Investigation of extraction solvent
Test solutions were prepared with different extraction solvents, respectively, and the measurement was performed according to the method described under item "3.1" above. As shown in FIG. 4 and Table 4, the results show that when water is used as the extraction solvent, the sample has no obvious characteristic peak in the chromatogram, so that the sample is excluded; when methanol and ethanol are used as solvents, the number of main chromatographic peaks of the sample solution is consistent, the peak shape difference of different extraction solvents is larger, and the extraction effect of ethanol is better than that of methanol, so that the sample extraction solvent is ethanol.
TABLE 4 comparison of extraction solvents
3.2.4 Investigation of sample size
Test solutions were prepared in different amounts and were measured as described under "3.1" above. As shown in FIG. 5 and Table 5, the results show that the sample solutions with different sampling amounts have the same number of main chromatographic peaks, smaller peak shape difference and similar extraction effect, but when the sampling amount is 3.0g, the response value of each chromatographic peak is larger, and the overall chromatogram is clearer and more obvious, so that the sampling amount for preparing the selected sample solution is 3.0g.
TABLE 5 comparison of sample volumes
3.2.5 Determination of sample preparation method
In summary, the main parameters of the method for preparing the sample solution are determined as follows: grinding the materials, taking about 3.0g, placing into a conical flask with a plug, adding 10ml of ethanol, heating and refluxing for 30 minutes, cooling, shaking, filtering, and collecting the filtrate.
3.3 Feature map analysis method verification
3.3.1 Investigation of specificity
Maltodextrin was taken at about 3.0g and measured as under item "3.1" above. The results show that: the main characteristic peaks in the chromatogram of the sample are well separated, maltodextrin is free from interference, and the method has good specificity, and is shown in figure 6.
3.3.2 Repeatability test
About 3.0g of poria peel formula granule sample (batch number: 220401) is taken, 6 parts are taken, the measurement is carried out according to the method of item 3.1, and the sample is introduced into 6 needles for measurement. As shown in FIG. 7, the measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number. The characteristic spectrum has 8 common peaks, the pachymic acid A (peak 3) is used as a reference peak S1, the pachymic acid (peak 6) is used as a reference peak S2, the relative retention time and the relative peak area of the peak 2, the peak 4 and the peak S1 are calculated, the relative retention time and the relative peak area of the peak 5, the peak 7, the peak 8 and the peak S2 are calculated, and the RSD value is calculated. The calculation shows that the relative retention time RSD% value and the relative peak area RSD% value are all within the acceptable range. Experiments show that the method has good repeatability, and the details are shown in tables 6-7.
TABLE 6 repeatability test characteristic spectra versus retention time
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 2 | 0.969 | 0.969 | 0.969 | 0.969 | 0.969 | 0.969 | 0.01 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 1.218 | 1.219 | 1.218 | 1.217 | 1.216 | 1.217 | 0.08 |
| 5 | 0.906 | 0.906 | 0.906 | 0.906 | 0.906 | 0.906 | 0.03 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.163 | 1.163 | 1.163 | 1.162 | 1.162 | 1.162 | 0.04 |
| 8 | 1.206 | 1.206 | 1.206 | 1.205 | 1.205 | 1.205 | 0.05 |
TABLE 7 repeatability test characteristic spectra versus peak area
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 2 | 0.264 | 0.264 | 0.262 | 0.268 | 0.268 | 0.269 | 0.99 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 0.251 | 0.253 | 0.256 | 0.255 | 0.257 | 0.258 | 1.05 |
| 5 | 0.481 | 0.482 | 0.484 | 0.492 | 0.481 | 0.487 | 0.85 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 0.993 | 0.965 | 1.009 | 0.977 | 0.986 | 0.998 | 1.59 |
| 8 | 0.891 | 0.884 | 0.918 | 0.918 | 0.904 | 0.915 | 1.61 |
3.3.3 Precision test
About 3.0g of poria peel formula particle (batch number: 220401) is taken and measured by continuous injection of 6 needles according to the test method under item 3.1. And 6-needle measurement is carried out by continuous sample injection. And 6-needle measurement is carried out by continuous sample injection. As shown in FIG. 8, the measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number. The characteristic spectrum has 8 common peaks, the pachymic acid A (peak 3) is used as a reference peak S1, the pachymic acid (peak 6) is used as a reference peak S2, the relative retention time and the relative peak area of the peak 2, the peak 4 and the peak S1 are calculated, the relative retention time and the relative peak area of the peak 5, the peak 7, the peak 8 and the peak S2 are calculated, and the RSD value is calculated. The calculation shows that the relative retention time RSD% value and the relative peak area RSD% value are all within the acceptable range. The calculation shows that the relative retention time RSD% value and the relative peak area RSD% value are all within the acceptable range. Experiments show that the method has good precision and is shown in tables 8-9.
TABLE 8 precision test HPLC characterization Profile versus Retention time
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 2 | 0.969 | 0.969 | 0.969 | 0.969 | 0.969 | 0.969 | 0.01 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 1.217 | 1.218 | 1.218 | 1.218 | 1.219 | 1.219 | 0.04 |
| 5 | 0.906 | 0.906 | 0.906 | 0.906 | 0.906 | 0.906 | 0.02 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.162 | 1.163 | 1.163 | 1.163 | 1.163 | 1.163 | 0.02 |
| 8 | 1.206 | 1.206 | 1.206 | 1.206 | 1.206 | 1.206 | 0.01 |
TABLE 9 precision test HPLC characteristic spectrum relative peak area
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 2 | 0.260 | 0.258 | 0.257 | 0.256 | 0.256 | 0.257 | 0.63 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 0.253 | 0.254 | 0.254 | 0.253 | 0.250 | 0.253 | 0.59 |
| 5 | 0.504 | 0.478 | 0.485 | 0.484 | 0.487 | 0.490 | 1.80 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.000 | 0.983 | 0.991 | 1.009 | 1.000 | 0.985 | 1.00 |
| 8 | 0.891 | 0.882 | 0.876 | 0.895 | 0.889 | 0.880 | 0.81 |
3.3.4 Stability test
About 3.0g of poria peel formula particles (batch number: 220401) are taken and are respectively sampled and measured at 0h, 2h, 4h, 8h, 12h and 24h according to the test method under the item "3.1". Sample injection measurement is carried out at 0h, 2h, 4h, 8h, 12h and 24h respectively. The measurement result shows that the peak shape and the peak number of the characteristic spectrum of the test sample are basically consistent. The characteristic spectrum has 8 common peaks, the pachymic acid A (peak 3) is used as a reference peak S1, the pachymic acid (peak 6) is used as a reference peak S2, the relative retention time and the relative peak area of the peak 2, the peak 4 and the peak S1 are calculated, the relative retention time and the relative peak area of the peak 5, the peak 7, the peak 8 and the peak S2 are calculated, and the RSD value is calculated. The calculation shows that the relative retention time RSD% value and the relative peak area RSD% value are all within the acceptable range. The test shows that the test solution is stable within 24 hours, see in detail fig. 9 and tables 10-11.
TABLE 10 stability test characterization profile versus retention time
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 2 | 0.969 | 0.969 | 0.969 | 0.969 | 0.969 | 0.969 | 0.01 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 1.217 | 1.218 | 1.218 | 1.219 | 1.218 | 1.217 | 0.06 |
| 5 | 0.906 | 0.906 | 0.906 | 0.906 | 0.906 | 0.906 | 0.02 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.162 | 1.163 | 1.163 | 1.163 | 1.163 | 1.162 | 0.03 |
| 8 | 1.206 | 1.206 | 1.206 | 1.206 | 1.206 | 1.205 | 0.03 |
TABLE 11 stability test characteristic spectrum versus peak area
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 2 | 0.260 | 0.258 | 0.257 | 0.256 | 0.255 | 0.270 | 2.07 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 0.253 | 0.254 | 0.254 | 0.250 | 0.251 | 0.258 | 1.07 |
| 5 | 0.504 | 0.478 | 0.485 | 0.487 | 0.481 | 0.486 | 1.86 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.000 | 0.983 | 0.991 | 1.000 | 0.993 | 0.986 | 0.70 |
| 8 | 0.891 | 0.882 | 0.876 | 0.889 | 0.891 | 0.910 | 1.29 |
3.4 Durability inspection
3.4.1 Investigation of different flow Rate (+ -0.02 ml/min)
About 3.0g of poria peel formula particle (batch number: 220401) is taken and the sample is introduced and measured according to the test method under item 3.1 at the flow rate of 0.98ml/min, 1.00ml/min and 1.02ml/min respectively. As shown in FIG. 12, the flow rates were 0.78ml/min, 0.80ml/min, and 0.82ml/min, respectively. The measurement result shows that the peak shape and the peak number of the characteristic spectrum of the test sample are basically consistent. The characteristic spectrum has 8 common peaks, the pachymic acid A (peak 3) is used as a reference peak S1, the pachymic acid (peak 6) is used as a reference peak S2, the relative retention time and the relative peak area of the peak 2, the peak 4 and the peak S1 are calculated, the relative retention time and the relative peak area of the peak 5, the peak 7, the peak 8 and the peak S2 are calculated, and the RSD value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are all within the acceptable range. The experiments show that the fine flow velocity change has less influence on the characteristic spectrum measurement, and the details are shown in tables 12-13.
TABLE 12 characterization by HPLC at various flow rates (+ -0.02 ml/min) versus retention time
| Peak number | 0.78ml/min | 0.80ml/min | 0.82ml/min | RSD％ |
| 2 | 0.963 | 0.963 | 0.962 | 0.08 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 1.213 | 1.216 | 1.219 | 0.21 |
| 5 | 0.905 | 0.904 | 0.903 | 0.11 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.163 | 1.164 | 1.164 | 0.04 |
| 8 | 1.206 | 1.207 | 1.208 | 0.09 |
TABLE 13 HPLC characterization profiles versus peak area for different flow Rate experiments (+ -0.02 ml/min)
| Peak number | 0.78ml/min | 0.80ml/min | 0.82ml/min | RSD％ |
| 2 | 0.270 | 0.269 | 0.273 | 0.71 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 0.250 | 0.250 | 0.252 | 0.47 |
| 5 | 0.483 | 0.491 | 0.499 | 1.67 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 0.978 | 0.988 | 1.006 | 1.48 |
| 8 | 0.929 | 0.936 | 0.942 | 0.74 |
3.4.2 Investigation of different flow rates (+ -0.2 ml/min)
About 3.0g of poria peel formula particle (batch number: 220401) is taken and the sample is introduced and measured according to the test method under item 3.1 at the flow rate of 0.60ml/min, 0.80ml/min and 1.00ml/min respectively. As shown in fig. 13, the measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number. The characteristic spectrum has 8 common peaks, the pachymic acid A (peak 3) is used as a reference peak S1, the pachymic acid (peak 6) is used as a reference peak S2, the relative retention time and the relative peak area of the peak 2, the peak 4 and the peak S1 are calculated, the relative retention time and the relative peak area of the peak 5, the peak 7, the peak 8 and the peak S2 are calculated, and the RSD value is calculated. The calculation shows that the relative retention time RSD% value, relative peak area RSD% value, are not within the acceptable range, see in detail tables 14-15. The test shows that the flow rate change of +/-0.2 ml/min has a great influence on the characteristic spectrum measurement.
TABLE 14 different flow rates (+ -0.2 ml/min) test HPLC profiles versus retention time
| Peak number | 0.60ml/min | 0.80ml/min | 1.00ml/min | RSD％ |
| 2 | 0.972 | 0.963 | 0.955 | 0.90 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 1.190 | 1.216 | 1.241 | 2.13 |
| 5 | 0.916 | 0.904 | 0.895 | 1.20 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 1.157 | 1.164 | 1.171 | 0.58 |
| 8 | 1.194 | 1.207 | 1.219 | 1.04 |
TABLE 15 HPLC characteristic profiles versus peak area for different flow rates (+ -0.2 ml/min) experiments
| Peak number | 0.60ml/min | 0.80ml/min | 1.00ml/min | RSD％ |
| 2 | 0.249 | 0.269 | 0.306 | 10.34 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 0.097 | 0.250 | 0.260 | 45.31 |
| 5 | 0.473 | 0.491 | 0.469 | 2.39 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 0.00 |
| 7 | 0.967 | 0.988 | 1.042 | 3.89 |
| 8 | 0.933 | 0.936 | 0.988 | 3.23 |
3.5 Knots
The characteristic spectrum method meets the requirements through investigation of specificity, precision, repeatability, stability and durability, and the established method is proved to be well used for characteristic spectrum measurement of poria cocos peel formula particles.
3.6 Characterization analysis of three batches of finished product feature patterns
According to the characteristic spectrum analysis method set forth in the above 3.1, three batches of finished product characteristic spectrum are measured, and the three batches of finished product characteristic spectrum are positioned by using the reference substances of pachymic acid A (concentration: 21.99 mug/ml), pachymic acid B (concentration: 21.48 mug/ml) and pachymic acid (concentration: 82.17 mug/ml). The results show that there are 8 common peaks in the three batches of finished product characteristic chromatograms, and the retention time of the 8 characteristic peaks in the chromatogram of the reference substance of the control medicinal material corresponds to that of the reference substances of the pachymic acid B, the pachymic acid A and the pachymic acid, wherein the corresponding peaks correspond to peak 1, peak 3 and peak 6, and are shown in figures 14-16.
Referring to fig. 15 and 16, it can be seen that 8 characteristic peaks should be present in the final product characteristic chromatogram and correspond to 8 characteristic peak retention times in the reference chromatogram of the reference medicinal material, wherein peak 1, peak 3, and peak 6 correspond to the reference peak retention times of pachymic acid B, pachymic acid a, and pachymic acid reference respectively. Specifically, the peak corresponding to the pachymic acid a reference peak is the S1 peak, the relative retention time of the peak 2, the peak 4 and the S1 peak is calculated, the relative retention time is within the range of + -10% of the specified value, and the specified value is: 0.96 (Peak 2), 1.21 (Peak 4); the peak corresponding to the pachymic acid reference peak is the S2 peak, the relative retention time of the peak 5, the peak 7, the peak 8 and the S2 peak is calculated, the relative retention time is within + -10% of the specified value, and the specified value is: 0.91 (Peak 5), 1.16 (Peak 7), 1.21 (Peak 8), and the ratio of the sum of Peak 1 Peak area and Peak 3 Peak area to Peak 6 Peak area should be greater than 10.
And (3) determining three batches of finished product characteristic patterns according to the drawn characteristic pattern analysis method. The results showed that there was 8 common peaks in the feature pattern, with pachymic acid a (peak 3) as reference peak S1 and pachymic acid (peak 6) as reference peak S2, the relative retention times of peaks 2, 4 and S1 were calculated, and the relative retention times and ranges of peaks 5, 7, 8 and S2 were calculated as detailed in table 16.
TABLE 16 relative retention time of common peaks for three batches
| Peak number | S16 | S17 | S18 | Mean value of | Relative retention time range.+ -. 10% |
| 2 | 0.957 | 0.957 | 0.957 | 0.957 | 0.861-1.052 |
| 3(S1) | 1.000 | 1.000 | 1.000 | 1.000 | / |
| 4 | 1.209 | 1.210 | 1.209 | 1.209 | 1.088-1.330 |
| 5 | 0.905 | 0.905 | 0.905 | 0.905 | 0.815-0.996 |
| 6(S2) | 1.000 | 1.000 | 1.000 | 1.000 | / |
| 7 | 1.162 | 1.163 | 1.163 | 1.163 | 1.046-1.279 |
| 8 | 1.206 | 1.207 | 1.207 | 1.207 | 1.086-1.327 |
In summary, the method for determining the characteristic spectrum of the formula particle established by adopting the high performance liquid chromatography performs stability investigation on the established method according to the four-part analysis method verification guidelines (general rule 9101) of the Chinese pharmacopoeia 2020 edition, and meets the requirements. According to the proposed characteristic spectrum analysis method, measuring characteristic spectrums of three batches of finished products, analyzing the result, calibrating 8 common characteristic peaks, calculating relative retention time of peak 2, peak 4 and S1 peak, calculating relative retention time of peak 5, peak 7, peak 8 and S2 peak, and respectively setting the average value of the relative retention time of the three batches of sample peaks as a specified value to be: 0.96 (Peak 2), 1.21 (Peak 4), 0.91 (Peak 5), 1.16 (Peak 7), 1.21 (Peak 8). The allowable range of the relative retention time is set to be +/-10% by considering multi-factor errors of test operation, instruments, reagents and the like.
4 Extract
4.1 Test methods
The extraction solvent of the traditional Chinese medicine formula granule is water for pharmacy, ethanol is used as solvent according to the technical requirement, and the hot dipping method under the alcohol-soluble extract measuring method (2201 in the general rule of the year 2020 of Chinese pharmacopoeia) is adopted, and the influence of auxiliary materials is simultaneously examined.
4.2 Investigation of extract
Taking about 2g of 3 batches of finished product samples produced on a large scale; the results are shown in Table 17.
TABLE 17 results of extract investigation
4.3 Extract Range determination
And (3) planning a scheme I:
and according to the specification of the product and the extract yield and the extract limit of the standard decoction, combining the extract measurement results of three batches of mass production samples, and setting the extract limit.
The specification of the product is that each 1g of particles is equivalent to 15g of decoction pieces, and the dry paste content of each 1g of particles is not less than 15g multiplied by 3.4% (namely, 0.51 g) according to the lower limit of the actual measurement range (3.4% -6.0%) of the paste yield of the standard decoction of the product; based on the extract limit (40.0%) of the standard decoction, it can be calculated that the extract content of each 1g granule is not less than 0.51gX40.0% (i.e. 0.204 g), or the extract content of the product (granule) is not less than 20.4% (0.204×100%).
The extract measurement values of the three batches of mass production samples of the product are respectively 43.5%, 44.0% and 43.2%, which are far higher than the calculated particle extract limit (20.4%), so the extract limit of the product is considered to be properly improved. For this purpose, it is assumed that the alcohol-soluble extract of the product should be not less than 28.0% calculated as 70% of the extract limit (40.0%) established in the standard decoction, i.e., 40.0% x 0.7=28.0%.
And (3) a second planning scheme:
The data for conversion of 28 batches of standard decoction extract of poria peel to granule extract are summarized in table 18:
table 18-28 conversion of Poria skin Label decoction into particulate extract summary table
Combining the paste yield, converting the extract of 28 batches of standard decoction samples into a granular extract according to the formulated product specification of 15g of decoction pieces per 1g of formula granule, wherein the average extract is 38.22%, the range of the measured value converted into the granular extract is 28.76-51.77%, and the SD is 5.72; the allowable range of the extract is 26.75 to 49.68 percent according to 70 to 130 percent of the average value. The allowable range of the extract is 21.05-55.39 percent calculated according to the average value of-3 SD to the average value of +3 SD. The allowed range of extract is assumed to be: the alcohol soluble extract of the product is not less than 26.8%.
Review of the above two extract limit considerations, comprehensive comparison, combined with three batch of mass production sample content data, ultimately formulated extract limit ranges of no less than 28.0%.
5 Content determination
5.1 Selection basis of the components to be measured
The exodermis Poria is dried exodermis of Polyporaceae fungus Poria cortex, poria cocos (Schw.) Wolf. It is produced in Anhui, hunan, hubei, henan and Yunnan. In addition, guizhou, sichuan, guangxi, fujian, zhejiang, hebei and the like. The products of Yunnan and Hunan have better quality and larger yield of Anhui and Hubei. The triterpenes in the skin include pachymic acid, dehydropachymic acid, pachymic acid A, pachymic acid B, etc. The pachymic acid A and the pachymic acid B are one of main active components of the pachymic skin, and occupy a larger proportion in the pachymic skin triterpene compounds, and according to the modern pharmacological research, the pachymic acid A and the pachymic acid B are widely applied in the aspects of anti-tumor, anti-inflammatory activity, immunoregulation and the like. Therefore, the research establishes the content methods of the pachymic acid A and the pachymic acid B to have important reference value for controlling the quality of the pachymic skin standard decoction. In the published province standard, the standard content measurement of tuckahoe formula particles in Guangdong, guizhou, guangxi and Shandong provinces uses tuckahoe acid B and tuckahoe acid A as content indexes.
5.2 Method for determining the content of Fuling acid B and Fuling acid A
The measurement is carried out by high performance liquid chromatography (the rule 0512 of the edition of Chinese pharmacopoeia 2020).
Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica as filler (4.6 mm. Times.250 mm,5 μm)); acetonitrile-0.1% formic acid (59:41) solution is taken as a mobile phase; the column temperature is 35 ℃; the detection wavelength was 252nm. The theoretical plate number is not less than 10000 calculated according to the pachymic acid A peak.
Preparation of a control solution: taking appropriate amounts of pachymic acid A and pachymic acid B, precisely weighing, and adding methanol to obtain mixed solution containing 100 μg pachymic acid A and 50 μg pachymic acid B per 1 ml.
Preparation of test solution: grinding the product, taking about 3.0g, precisely weighing, placing into a conical flask with a plug, precisely adding 10ml of ethanol, sealing, weighing, performing ultrasonic treatment (power 300W, frequency 50 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with ethanol, shaking, filtering, and collecting the subsequent filtrate.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
5.3 Pretreatment investigation of the test sample for determining the total content of Fuling acid A and Fuling acid B
The extraction mode, the extraction solvent, the extraction time and the sampling amount (or the amount of the solvent) of the poria cocos formula particle (batch number: 220401) content measurement are examined to determine the optimal sample pretreatment mode.
5.3.1 Investigation of the extraction method
The sample preparation test solutions were extracted in different ways and assayed according to the method described under "5.2" above. The results show that the reflux extraction is superior to the ultrasonic extraction, so the sample extraction mode is chosen as reflux extraction, and the details are shown in Table 19.
TABLE 19 comparison of different extraction methods
5.3.2 Investigation of extraction time
According to the method described in item "5.2", the sample is extracted at different extraction times to prepare a sample solution, and the measurement is performed according to law. The results show that the ultrasonic extraction can be basically complete within 30 minutes, so the sample extraction time is determined to be 30 minutes, and the details are shown in Table 24.
TABLE 20 comparison of different extraction times
5.3.3 Investigation of extraction solvent
According to the method described in item "5.2", the sample solutions were prepared by extracting the samples with different solvents, respectively, and the measurement was carried out according to the law. The result shows that when water is used as a solvent, no obvious characteristic peak exists in the chromatogram of the test sample, so that the test sample is eliminated; the extraction content difference is larger when methanol and ethanol are used as solvents, so ethanol with smaller toxicity and higher content is selected as the solvent for preparing the test sample solution, and the details are shown in table 21.
TABLE 21 comparison of different extraction solvents
5.3.4 Sample size investigation
Test solutions were prepared in different amounts by the method described under "5.2" above, and the measurement was performed according to the law. As a result, the extraction contents of the different sampling amounts were not much different, and the chromatographic peak response was higher as compared with 1.0g and 1.75g, so that 3.0g was selected as the sampling amount for preparing the sample solution, as shown in Table 22.
TABLE 22 comparison of different sample volumes
In summary, the main parameters of the test solution preparation method are determined as follows: taking a proper amount of the product, grinding, taking about 3.0g, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of ethanol, weighing, heating and refluxing for 30 minutes, cooling, weighing again, supplementing the weight loss with ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
5.4 Content determination analysis method verification
5.4.1 Specificity investigation
Maltodextrin was taken at about 3.0g and a maltodextrin blank was prepared as described under item "5.2" above. As shown in fig. 17, the test showed that: the maltodextrin has no interference to blank, and the method has good specificity.
5.4.2 Linear relationship test
(1) Taking a poria cocos acid A reference substance solution with the concentration: 1.1795. Mu.g/ml, 11.7946. Mu.g/ml, 23.5893. Mu.g/ml, 47.1785. Mu.g/ml, 94.3570. Mu.g/ml, 188.7140. Mu.g/ml were determined by chromatographic conditions under item "5.2". Drawing a standard curve by taking the sample injection amount of the pachymic acid A as an abscissa and the peak area as an ordinate, performing linear regression,
The regression equation is: y=1E-06x+0.0039r 2 =0.9999
It can be seen that the pachymic acid A has a good linear relationship with its peak area in the range of 1.1795. Mu.g/ml to 188.7140. Mu.g/ml, as shown in FIG. 18 and Table 23.
TABLE 23 Linearity examination results of Functions acid A
| Numbering device | Peak area | Sample injection amount mug |
| Linearity 1 | 10347.0 | 0.0118 |
| Linearity 2 | 120966 | 0.1179 |
| Linearity 3 | 239196 | 0.2359 |
| Linearity 4 | 480725 | 0.4718 |
| Linearity 5 | 926924 | 0.9436 |
| Linearity 6 | 1871161 | 1.8871 |
(2) Taking a poria cocos acid B reference substance solution with the concentration: 1.0204. Mu.g/ml, 10.2043. Mu.g/ml, 20.4085. Mu.g/ml, 40.8170. Mu.g/ml, 81.6340. Mu.g/ml, 163.2680. Mu.g/ml were determined by chromatographic conditions under item "5.2". And drawing a standard curve by taking the sample injection amount of the pachymic acid B as an abscissa and the peak area as an ordinate, and performing linear regression.
The regression equation is: y=9E-07x+0.0019r 2 =1.000
It can be seen that the pachymic acid B has a good linear relationship with its peak area in the range of 1.0204. Mu.g/ml to 163.2680. Mu.g/ml, as shown in FIG. 19 and Table 24.
TABLE 24 Linearity examination results of Functions acid B
5.4.3 Repeatability test
About 3.0g of poria cocos peel formula particle samples (batch number: 220401) in the same batch are taken, 6 parts are taken, the poria cocos peel formula particle samples are measured according to the test method under the item "5.2", the average value of the content of the poria cocos acids in the samples is 9.0791mg/g, the RSD value is 0.95%, the average value of the total content of the poria cocos acids A and the poria cocos acids B in the samples is 0.5249mg/g, the RSD value is 0.43%, and the test shows that the method is good in repeatability, and the details are shown in Table 25.
Table 25 repeatability test
5.4.4 Precision test
About 3.0g of poria cocos peel formula particle sample (batch number: 220401) is taken, 6 needles are continuously injected according to the method under the item "5.2", the peak area is measured, the RSD value of the total content of the poria cocos acid A and the poria cocos acid B in the sample is calculated to be 0.17%, and the test shows that the instrument precision is good, and the details are shown in Table 26.
TABLE 26 precision test
5.4.5 Stability test
About 3.0g of poria cocos peel formula particle sample (220401) is taken, samples are respectively taken at 0h, 2h, 4h, 8h, 12h and 24h according to the test method under the item "5.2", the content of the poria cocos peel formula particle sample is measured, the RSD value of the total content of the poria cocos acid A and the poria cocos acid B in the sample is calculated to be 0.40%, and the test shows that the test sample solution is stable within 24 hours, and the details are shown in Table 27.
TABLE 27 stability test
5.4.6 Sample recovery test
About 1.50g of poria cocos peel formula particle samples (batch number: 220401) are taken, 9 parts are precisely weighed and divided into 3 groups, 3 parts of each group are respectively added with 2.4ml of poria cocos acid A (179.8284 mug/ml) control, 3.0ml, 3.5ml of poria cocos acid B (171.8284 mug/ml) control, 1.2ml, 1.4ml and 1.6ml of poria cocos acid A (171.8284 mug/ml) control with known concentration, test solution is prepared according to the method under item "5.2", measurement is carried out, and the average sample adding recovery rate of the total content of the poria cocos acid A and the poria cocos acid B is calculated to be 189.96%, and RSD is 1.04%, and is shown in a table 28 in detail.
TABLE 28 sample recovery test results
5.5 Knots
In conclusion, the whole analysis method meets the requirements through specificity, peak purity, precision, repeatability, stability, linear investigation and sample addition recovery tests, and the results show that the established method can be well used for content determination of the total content of the pachymic acid A and the pachymic acid B.
5.6 Determination of the content of three batches of finished products
The total content of the finished products of pachymic acid A and pachymic acid B in three batches was determined according to the above-mentioned formulated content analysis method, and the results are shown in Table 29.
TABLE 29 determination of three batch finished product
5.7 Content Limit Range determination
(1) Content range planning scheme one:
According to the specification of the product and the control range of the paste yield and the content of the standard decoction, the content limit range is formulated by combining the measurement results of three batches of mass production samples.
The specification of the product is that each 1g of particles is equivalent to 15g of decoction pieces, and the dry paste content of each 1g of particles is 15g multiplied by 3.0% -15 g multiplied by 6.0% (namely 0.45 mg-0.90 mg), the auxiliary material content is not more than 0.50mg, and the lower limit is 0.50 mg) according to the allowable range (3.0% -6.0%) of the paste yield of the standard decoction of the product; based on the above, the allowable range of the total content of the pachymic acid A and the pachymic acid B (0.34 mg/g to 1.60 mg/g) can be calculated according to the allowable range of the total content of the pachymic acid A and the pachymic acid B (0.50 multiplied by 0.34mg/g to 0.90 multiplied by 1.60 mg/g) (namely 0.2mg to 1.4 mg) in each 1g particle. The contents of three batches of mass production samples of the product are respectively 0.52mg/g, 0.54mg/g and 0.50mg/g, and the contents of the particles calculated or planned based on the standard decoction are all within the range.
In conclusion, the total content range of the poria cocos acid A and the poria cocos acid B of the product is 0.2 mg/g-1.4 mg/g.
(2) Content range planning scheme II:
the content of the poria peel standard decoction is converted into the content of granules according to 28 batches of poria peel standard decoction, and the contents are summarized as follows:
Table 30-28 conversion of Poria cocos skin Standard decoction into particle content summary table
Combining the paste yield, converting the content of 28 batches of standard decoction samples into particle content according to the specification of the formulated product, wherein each 1g of formula particles is equivalent to 15g of decoction pieces, the average value of the total content of the pachymic acid A and the pachymic acid B is 0.661mg/g, the measured content is converted into the particle content range of 0.35mg/g to 1.12mg/g, and the SD is 0.177; calculated according to 70-130% of the average value, the allowable range of the total content of the pachymic acid A and the pachymic acid B is 0.46 mg/g-0.86 mg/g. The total content allowable range of the pachymic acid A and the pachymic acid B is 0.129mg/g to 1.193mg/g calculated according to the average value of-3 SD to the average value of +3 SD. For this purpose, the allowable range of the total content of the pachymic acid A and the pachymic acid B is 0.13mg/g to 1.19mg/g (average value + -3SD is rounded).
For review, the two content ranges are considered, comprehensively compared, and the total content range of the pachymic acid A and the pachymic acid B is 0.2mg/g to 1.4mg/g by combining the content data of three batches of mass production samples.
6 Comparative study of Poria skin and Poria
6.1 Comparing the pachymaran with pachymaran A, pachymaran B and pachymaran peak areas in the standard decoction of pachymaran, see tables 31 and 32.
Table 31-28 Fuling acid a, fuling acid B and Fuling acid peak area comparison summary table in Fuling skin Standard decoction
Table 32-26 Fuling Standard decoction Fuling acid A, fuling acid B and Fuling acid Peak area comparison summary table
In summary, the relative peak areas of the pachymic acid A and the pachymic acid in the pachymic acid standard soup are all smaller than 1.0, the relative peak areas of the pachymic acid B and the pachymic acid are all smaller than 1.5, and the ratio of the sum of the pachymic acid A and the pachymic acid B to the pachymic acid peak area is all smaller than 2.0; the relative peak areas of the pachymic acid A and the pachymic acid in the pachymic acid skin standard soup are both larger than 6.0, the relative peak areas of the pachymic acid B and the pachymic acid are both larger than 4.0, and the ratio of the sum of the peak areas of the pachymic acid A and the pachymic acid B to the peak area of the pachymic acid is both larger than 10.0.
6.2 Comparing the areas of Fuling acid A, fuling acid B and Fuling acid peaks in the finished product of Fuling Pi and Fuling formula granule, see tables 33 and 34.
TABLE 33-3 Fuling skin formulas Fuling acid A, fuling acid B and Fuling acid Peak area vs. summary table
Table 34-3 batch Poria cocos formula granule Fuling acid a, fuling acid B and Fuling acid peak area comparison summary table
As can be known from the comparison, the content of the pachymic acid A and the pachymic acid B in the pachymic skin is far higher than the content of the pachymic skin, the ratio of the (pachymic acid A+pachymic acid B)/the pachymic acid is higher than 10, the ratio of the pachymic acid is lower than 2.5, and the ratio of the index is clearly required in the quality standard, so that the pachymic acid formula particle or the pachymic skin formula particle is distinguished, two products can be prevented from being distinguished in production and clinical application, and counterfeit and inferior products are prevented.
The 7 specifications are formulated according to the preparation method feeding amount and the process research making amount, namely each 1g of formula particle is equivalent to 15g of decoction pieces.
8, The storage is formulated according to the basic requirements of variety storage.
Those skilled in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for detecting the quality of poria peel formula particles is characterized by comprising the following detection methods,
Determining the properties, dry extract yield, thin layer identification, characteristic patterns, extract and content of pachymic acid A and pachymic acid B of the pachymic skin formula particles, and limiting the content standard of the pachymic skin formula particles to be 0.2 mg/g-1.4 mg/g of total content of pachymic acid A and pachymic acid B, wherein the thin layer identification adopts thin layer chromatography for identification; the characteristic spectrum and the content measurement of the pachymic acid A and the pachymic acid B are all measured by adopting a liquid chromatography; the extract is measured by a hot dipping method;
the determination of the characteristic spectrum by liquid chromatography comprises: analyzing with liquid chromatograph, taking the solution prepared from Poria skin control medicinal material as reference material solution B, taking the solutions prepared from Poria acid A and Poria acid B reference material as reference mixed solution B, taking the solution prepared from Poria skin granule sample as test material solution B, respectively precisely sucking the reference material solution B, reference mixed solution B and test material solution B, respectively injecting into liquid chromatograph, and measuring to obtain the final product; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica is used as a filler, shimadzu Shim-PACK GIST C-AQ (4.6 mm. Times.250 mm,5 μm); mobile phase: acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table a;
Table a gradient elution procedure
Flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: the time period of 0 to 53 minutes is 242nm, and the time period of 53 to 105 minutes is 210nm.
2. The method for detecting the quality of poria peel formula particles according to claim 1, wherein the poria peel formula particles are prepared by the following steps: decocting Poria cortex decoction pieces 15000g in water, filtering, concentrating the filtrate into fluid extract, adding appropriate amount of adjuvant, drying, adding appropriate amount of adjuvant, mixing, granulating, and making into 1000 g.
3. The method for detecting the quality of poria peel formula granules according to claim 1, wherein the thin-layer chromatography comprises the following steps:
(1) Preparing a test sample solution a: taking 5.0g of poria cocos peel formula particle sample, grinding, adding 50ml of diethyl ether, carrying out ultrasonic treatment for 10 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution a;
(2) Preparing a control medicinal material solution a: taking 0.5g of poria peel reference medicinal material, grinding, adding 50ml of diethyl ether, carrying out ultrasonic treatment for 10 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a reference medicinal material solution a;
(3) Thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel H thin layer plate; sample application amount: the solution a of the sample is 10uL, the solution a of the reference medicinal material is 1uL, and the solution a and the reference medicinal material are respectively spotted on the same silica gel H thin layer plate; toluene-ethyl acetate-formic acid (20:5:0.5) solution is taken as a developing agent; color-developing agent: 2% vanillin sulfuric acid solution-ethanol (4:1) mixture was heated at 105℃and inspected in sunlight.
4. The method for detecting the quality of poria peel formula granules according to claim 1, wherein the hot dipping method uses ethanol as a solvent, and the range of the extract is measured by adopting a hot dipping method under the alcohol-soluble extract measuring method.
5. The method for detecting the quality of poria peel formula particles according to claim 1, wherein the characteristic spectrum is measured by liquid chromatography, further comprising the steps of:
(1) Preparing a reference substance solution b of a control medicinal material: taking 1.0g of poria peel reference medicinal material, adding 10ml of ethanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, and taking a subsequent filtrate as a reference substance solution b of the reference medicinal material;
(2) Preparing a reference substance mixed solution b: taking a proper amount of reference substances of pachymic acid, pachymic acid A and pachymic acid B, precisely weighing, and adding methanol to prepare reference substance mixed solution B containing 80 mug of pachymic acid, 20 mug of pachymic acid A and 20 mug of pachymic acid B per 1ml respectively;
(3) Preparing a test sample solution b: taking proper amount of poria peel formula particles, grinding, taking about 3.0g, placing into a conical flask with a plug, adding 10ml of ethanol, sealing, performing ultrasonic treatment for 30 minutes, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution b.
6. The method for detecting the quality of poria peel formula particles according to claim 1, wherein the liquid chromatography is used for determining poria peel formula particles, 8 characteristic peaks are required to be displayed in a sample chromatograph obtained, the 8 characteristic peaks are required to correspond to 8 characteristic peak retention times in a reference chromatograph of a reference medicinal material, the sample chromatograph is respectively characterized by being respectively corresponding to a poria acid B reference peak, a poria acid a reference peak and a poria acid reference peak in peak 1, peak 3 and peak 6 of the poria acid, the peak corresponding to the poria acid a reference peak in the sample chromatograph is an S1 peak, the relative retention times of peak 2, peak 4 and the S1 peak are calculated, the relative retention times are within +/-10% of the specified values, and the specified values are: peak 2 is 0.96, peak 4 is 1.21, peak corresponding to pachymic acid reference peak is S2 peak, and relative retention time of peak 5, peak 7, peak 8 and S2 peak is calculated, wherein the relative retention time is within + -10% of the specified value, the specified value is: peak 5 is 0.91, peak 7 is 1.16, peak 8 is 1.21, and the ratio of the sum of peak 1 and peak 3 peak areas to peak 6 peak area should be required to be greater than 10.
7. The method for detecting the quality of poria cocos peel formula particles according to claim 1, wherein the determination of the content of poria cocos acids a and B by liquid chromatography comprises: performing liquid chromatograph analysis, taking solutions prepared from pachymic acid A and pachymic acid B as reference solutions c, taking solutions prepared from pachymic skin granule samples as test solutions c, precisely sucking the reference solutions c and the test solutions c respectively, respectively injecting into liquid chromatograph, and measuring to obtain the final product; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica as filler (4.6mm. Times.250 mm,5 μm); mobile phase: acetonitrile-0.1% formic acid (59:41) solution is taken as a mobile phase, and gradient elution is carried out according to the specification; column temperature: 35 ℃; sample injection amount: 10. Mu.L; detection wavelength: 252nm.
8. The method for detecting the quality of poria cocos peel formula particles according to claim 7, wherein the method for detecting the content of poria cocos acids A and B by liquid chromatography further comprises the following steps:
(1) Preparing a reference substance solution: taking a proper amount of a reference substance of the pachymic acid A and the pachymic acid B, precisely weighing, adding methanol to prepare a mixed solution containing 100 mug of the pachymic acid A and 50 mug of the pachymic acid B per 1ml, and taking the mixed solution as a reference substance solution c;
(2) Preparing a test solution: taking proper amount of poria peel formula particles, grinding, taking about 3.0g, precisely weighing, placing into a conical bottle with a plug, precisely adding 10ml of ethanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing loss weight with ethanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.
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