CN113791164B - Quality detection method for standard decoction of scalded rhizoma Cibotii - Google Patents
Quality detection method for standard decoction of scalded rhizoma Cibotii Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/95—Detectors specially adapted therefor; Signal analysis
Abstract
The application provides a quality detection method of a standard decoction of a hot rhizoma cibotii, which comprises the steps of determining the properties of the standard decoction of the hot rhizoma cibotii, the extract yield of a dry extract, thin-layer identification, extract, characteristic spectrum and protocatechuic acid content, limiting the standard decoction content standard to 0.80-5.20mg of protocatechuic acid in each 1g, wherein the extract yield of the dry extract is determined by a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the protocatechuic acid content are all measured by liquid chromatography. According to the method for detecting the quality of the standard decoction of the scalded rhizoma cibotii, disclosed by the application, the quality of the standard decoction of the scalded rhizoma cibotii is evaluated through multi-aspect measurement, a solid foundation is laid for the stability of the quality of a product, a feasible quality standard of the standard decoction of the scalded rhizoma cibotii can be established, and the effective control of the quality of the standard decoction of the scalded rhizoma cibotii is realized.
Description
Technical Field
The application relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality detection method of a standard decoction of a hot rhizoma cibotii.
Background
The modern medicine needs to have three characteristics of stability, uniformity, safety and effectiveness, and the Chinese patent medicine is difficult to compare with western medicines in the aspects, so that the detection is more needed by adopting various means, and the reliability and the stability of the detection result are ensured. The scalding rhizoma Cibotii is prepared from dried rhizome of rhizoma Cibotii Cibotium barometz (L.) J.Sm. Of the family Pteridaceae, and has effects of nourishing liver and kidney, eliminating rheumatism, strengthening waist and foot, benefiting joint, and treating waist and back soreness, knee pain, foot weakness, cold-dampness peri-arthralgia, drowning, frequent urination, spermatorrhea, and leucorrhea. At present, the detection method of the scalded rhizoma cibotii medicinal material mainly adopts a liquid chromatography, however, the detection of the scalded rhizoma cibotii decoction by adopting the existing liquid chromatography is defective, and the quality control requirement of the traditional Chinese medicine formula particles cannot be met. Therefore, it is necessary to establish a method for detecting the quality of the standard decoction of the scalded rhizoma Cibotii for controlling the quality of medicinal materials.
Disclosure of Invention
The application aims to solve the defects in the prior art and provide a method for detecting the quality of a standard decoction of a hot rhizoma cibotii, so as to better control the quality of the decoction of the hot rhizoma cibotii, characterize the quality of a medicament and improve the stability of the medicament.
In order to achieve the above purpose, the technical scheme of the application is realized as follows:
the application provides a method for detecting the quality of a standard decoction of a hot rhizoma cibotii, which comprises the following steps of detecting,
the standard decoction content standard is limited to 0.80-5.20mg of protocatechuic acid per 1g by measuring the characters of the standard decoction of the scalded rhizoma Cibotii, the extract yield of the dry extract, the thin-layer identification, the extract, the characteristic map and the protocatechuic acid content, wherein the extract yield of the dry extract is measured by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the protocatechuic acid content are measured by liquid chromatography;
the determination of the characteristic spectrum by liquid chromatography comprises: analyzing with liquid chromatograph, taking the solution prepared by scalding rhizoma Cibotii reference medicine as reference substance solution, the solution prepared by protocatechuic acid reference substance as reference substance solution, and the solution prepared by scalding rhizoma Cibotii standard decoction sample as test substance solution, respectively precisely sucking the reference substance solution, the reference substance solution and the test substance solution, respectively injecting into liquid chromatograph, and measuring to obtain the final product; wherein the chromatographic conditions used are C18 (250 mmx4.6mm,5 um) (Waters XSelec HSS T3); mobile phase: using methanol as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, and performing gradient elution according to the specification of a table a;
table a gradient elution procedure
Flow rate: 1.0mL/min; column temperature: 35 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm.
In one embodiment, the decoction method comprises: the decoction method comprises the following steps: soaking rhizoma Cibotii decoction pieces in water for 30-40min, decocting twice for 30-40min for the first time and 25-30min for the second time, separating solid from liquid while hot, mixing filtrates, concentrating, and drying to obtain rhizoma Cibotii standard decoction dry extract powder.
In one embodiment, the thin layer chromatography comprises the steps of:
(1) Preparing a test sample solution a: taking 2g of a standard decoction sample of the scalded rhizoma Cibotii, adding 50mL of methanol, carrying out ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, and dissolving residues with 1mL of methanol to prepare a sample solution a;
(2) Preparing a reference substance solution a: dissolving protocatechuic acid reference substance in methanol to obtain reference substance solution a with concentration of 1 mg/mL;
(3) Thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel G thin layer plate; sample application amount: 1uL of each of the sample solution a and the reference solution a; developing agent: chloroform-ethyl acetate-methanol-formic acid with a volume ratio of 12:2:1:0.8; color-developing agent: temporarily preparing 2% ferric trichloride solution-1% potassium ferricyanide solution, wherein the volume ratio of the 2% ferric trichloride solution to the 1% potassium ferricyanide solution is 1:1, and viewing under 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) Preparation of reference solution b: decocting rhizoma Cibotii control 1.0g in 25mL water for 1 hr, filtering, evaporating filtrate to dryness, adding 5mL methanol into the residue, filtering, and collecting filtrate as reference solution b;
(2) Preparing a reference substance solution b: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol for dissolving, and preparing a reference substance solution b with the concentration of 0.1 mg/mL;
(3) Preparing a test sample solution b: taking 1.0g of a standard decoction sample of the scalded rhizoma cibotii, precisely weighing, placing the standard decoction sample into a conical bottle with a plug, adding 25mL of precisely weighed methanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking filtrate as a sample solution b.
In one embodiment, the determination of protocatechuic acid content by liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from protocatechuic acid reference substance as a reference substance solution c, taking a solution prepared from a standard decoction sample of the hot rhizoma Cibotii as a sample solution c, precisely sucking the reference substance solution c and the sample solution c respectively, respectively injecting into the liquid chromatograph, and measuring to obtain the final product; wherein the chromatographic conditions used are C18 (250 mmx4.6mm,5 um) (Waters XSelec HSS T3); mobile phase: taking methanol as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, and performing gradient elution according to a specified rule; flow rate: 1.0mL/min; column temperature: 35 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm.
In one embodiment, the method for measuring the protocatechuic acid content by liquid chromatography further comprises the following steps:
(1) Preparing a reference substance solution: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol to prepare a solution with the concentration of the protocatechuic acid of 0.1mg/ml, and taking the solution as reference substance solution c;
(2) Preparing a test solution: taking about 1.0g of a standard decoction sample of the scalded rhizoma cibotii, precisely weighing, placing the standard decoction sample into a conical flask with a plug, precisely adding 25mL of methanol, sealing, weighing by ultrasonic for 30min, cooling, weighing again, supplementing the weight loss by using the methanol, shaking uniformly, and filtering to obtain a sample solution c.
Compared with the prior art, the application has the beneficial effects that:
(1) The quality of the standard decoction of the hot rhizoma cibotii is evaluated through various measurement by researching the properties of the standard decoction of the hot rhizoma cibotii, the dry extract extraction rate, the thin-layer identification, the extract, the characteristic spectrum and the protocatechuic acid content measurement, a solid foundation is laid for the stable quality of the product, the feasible quality standard of the decoction of the hot rhizoma cibotii can be established, the effective control of the quality of the standard decoction of the hot rhizoma cibotii is realized, and the chromatographic condition is adopted for liquid phase analysis, so that a chromatogram with better and clearer separation degree can be obtained.
(2) The standard decoction of the scalding rhizoma cibotii decoction pieces is prepared by a decoction method, the average content of protocatechuic acid is 1.92mg/g, the measured content range is 1.10-4.00mg/g, the SD (standard deviation) is 1.12, the allowable range of protocatechuic acid content is 1.35-2.50mg/g according to the average value of 70-130%, and the allowable range of protocatechuic acid content of the standard decoction is 0.80-5.28 mg/g according to the low limit of-SD and the high limit of-3 SD. The average transfer rate of protocatechuic acid is 64.02%, the transfer rate range is 37.8% -87.8%, SD is 19.43, according to the technical requirements of quality control and Standard establishment of traditional Chinese medicine prescription granule, the allowable range of protocatechuic acid content transfer rate is calculated according to 70% -130% of the transfer rate average value, and the allowable range of protocatechuic acid content transfer rate is 44.81-83.23%. The allowable range of the protocatechuic acid content transfer rate is 25.16 to 102.88 percent according to +/-2 SD. According to the data, the limit of measuring the content of protocatechuic acid in the standard decoction is calculated as follows: 0.80 mg/g-5.20 mg/g, and the allowable range of the content transfer rate is 25.0% -100.0%. The results show that the protocatechuic acid content and the transfer rate of the protocatechuic acid in the standard decoction of a plurality of batches are all within the allowable range, so the application can provide reference for the quality standard research of the scalded rhizoma cibotii formula particles.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a thin layer diagram of a standard decoction of 15 batches of decoction pieces of rhizoma Cibotii; wherein, the group 1 of the patterns is a negative control sample thin layer pattern, the group 2 is a protocatechuic acid control solution thin layer pattern, and the group 3-17 is a standard decoction thin layer pattern of 15 batches of the scalded rhizoma cibotii decoction pieces.
FIG. 2 is a comparison chart of different extraction methods in the investigation of the extraction method of the application; s1 is ultrasonic extraction of a characteristic spectrum of a sample solution; s2, reflux extraction of a characteristic spectrum of the sample solution.
FIG. 3 is a graph showing the comparison of different extraction solvents in the investigation of the extraction solvents of the present application; wherein S1 is a characteristic spectrum of a sample solution prepared by extracting 20% methanol; s2 is a characteristic spectrum of a sample solution prepared by extracting 50% ethanol; s3 is a characteristic spectrum of the sample solution prepared by methanol extraction.
FIG. 4 is a graph showing the comparison of different sample amounts in the investigation of the sample taking amount according to the present application; s1 is a characteristic spectrum of a sample solution to be tested, wherein the sample taking amount of the characteristic spectrum is 0.5 g; s2 is a characteristic spectrum of a sample solution to be tested, the sample taking amount of which is 1.0 g; s3 is a characteristic spectrum of the sample solution with the sample taking amount of 1.5 g.
FIG. 5 is a graph showing the comparison of different extraction times in the extraction time investigation of the present application; wherein S1 is a characteristic spectrum of a sample solution to be extracted for 20min by ultrasonic extraction; s2, ultrasonically extracting a characteristic spectrum of the sample solution for 30 minutes; s3, ultrasonic extraction is carried out for 40min on the characteristic spectrum of the sample solution.
FIG. 6 is a graph comparing blank solvents in the investigation of the specificity of the present application; wherein S1 is a blank solvent (methanol) characteristic map; s2 is a reference substance solution characteristic map; s3 is a characteristic spectrum of the solution of the test sample.
FIG. 7 is a graph of the common peak superposition characteristics of the repeatability test of the present application; s1 is a test sample solution common peak superposition characteristic spectrum under repeatability 1; s2, overlapping a characteristic spectrum by a common peak of the sample solution under the condition of the renaturation 2; s3 is a test sample solution shared peak superposition characteristic map under repeatability 3; s4 is a test sample solution shared peak superposition characteristic map under the condition of repeatability 4; s5, overlapping a characteristic spectrum by a common peak of the sample solution under the condition of the renaturation 5; s6, overlapping a characteristic spectrum by a common peak of the sample solution under the condition of the renaturation 6; s7 is a control map.
FIG. 8 is a graph of the common peak superposition characteristics for the precision test of the present application; s1 is a test sample solution common peak superposition characteristic spectrum under the precision of 1; s2 is a test sample solution common peak superposition characteristic spectrum under the precision of 2; s3 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 3; s4 is a test sample solution common peak superposition characteristic spectrum under the precision of 4; s5 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 5; s6, a test sample solution common peak superposition characteristic spectrum under the condition of precision 6; s7 is a control map.
FIG. 9 is a graph of the common peak superposition characteristics for the stability test of the present application; s1 is a test sample solution common peak superposition characteristic spectrum measured in 0 h; s2 is a test sample solution common peak superposition characteristic spectrum measured in 2 h; s3, a common peak superposition characteristic spectrum of the sample solution measured in 4 hours; s4, a common peak superposition characteristic spectrum of the sample solution measured in 8 hours; s5, a test sample solution common peak superposition characteristic spectrum measured in 12 hours; s6, a characteristic spectrum of the common peak superposition of the test sample solution measured in 24 hours; s7 is a control map.
FIG. 10 shows the characteristic spectrum of the standard decoction of the present application for determining the original catechin as reference.
FIG. 11 is a graph of a reference drug of the scalding rhizoma Cibotii in the measurement of the characteristic spectrum of the standard decoction of the application.
FIG. 12 is a superposition spectrum of 15 batches of hot rhizoma Cibotii traditional Chinese medicinal materials in the measurement of the characteristic spectrum of the standard decoction of the application; wherein S1-S15 represents a superposition map of 1-15 batches of hot rhizoma Cibotii Chinese medicinal materials.
FIG. 13 is a graph showing the superposition of characteristic maps of 15 batches of standard decoction, reference substances and reference medicinal materials according to the application; wherein S1 represents a reference substance characteristic spectrum, S2 represents a reference medicinal material characteristic spectrum, and S3-S17 represents a standard decoction superposition spectrum of 1-15 batches of scalding rhizoma Cibotii.
Fig. 14 is a graph showing a fit of 15 lot of standard decoction of the hot rhizoma Cibotii in the measurement of the characteristic spectrum of the standard decoction of the present application.
FIG. 15 is a graph showing the linearity of the concentration of protocatechuic acid control in the linear range test of the present application.
Detailed Description
The present application 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 application more apparent.
The application provides a quality detection method of a standard decoction of a hot rhizoma cibotii, which comprises the following detection method, wherein the standard decoction content standard is limited to 0.80-5.20mg of protocatechuic acid in each 1g by measuring the properties of the standard decoction of the hot rhizoma cibotii, the extract yield of a dry extract, thin-layer identification, extract, characteristic spectrum and protocatechuic acid content, and the extract yield of the dry extract is measured by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the protocatechuic acid content are all measured by liquid chromatography.
In this embodiment:
preparing a standard decoction of the hot rhizoma cibotii: referring to the decoction method in the management Specification of Chinese medicine decoction Chamber of medical institutions (No. 2009) of Chinese medicine administration, 15 batches of decoction pieces of the rhizoma Cibotii are taken, water is added until the decoction pieces of the rhizoma Cibotii are about 4-5cm higher than the medicinal materials, the decoction pieces are soaked for 30-40min, the decoction is carried out twice, the first decoction time is 30-40min, the second decoction time is 25-30min, solid-liquid separation is carried out while the decoction pieces are still hot, the filtrate is combined, concentrated and dried, and 15 batches of standard decoction dry paste powder of the rhizoma Cibotii are prepared.
1. Property investigation
According to the physical characteristics of 15 batches of the standard decoction of the hot rhizoma cibotii, the standard decoction is described as yellowish-brown to tan particles, and has light smell, light taste and slightly astringent taste.
2. Dry extract yield test
The 15 batches of the scalding rhizoma cibotii decoction pieces are taken, 15 batches of standard decoction dry paste powder are prepared according to the preparation method, dry extract yield is calculated according to the dry paste powder (see table 1), average yield is calculated to be 17.813%, and the allowable range of paste yield is calculated according to the allowable range of standard decoction paste yield (average value 70% -130%), so that the paste yield is calculated to be 12.47% -23.16%, and the paste yield is calculated to be 12.5% -23.0%.
Table 1: paste yield
The results show that the dry extract yield of 15 batches of standard decoction is 15.8-20.2%, and the dry extract yield accords with the range of 12.5-23.0% of the planned limit.
3. Thin layer authentication
The product of the application is a dried extract of single decoction piece scalding rhizoma cibotii, and the method under the item of scalding rhizoma cibotii thin layer identification in Chinese pharmacopoeia is referred to, a protocatechuic acid reference substance is used as a reference, the thin layer identification method is established, 15 batches of sample tests prove that spots of the test sample are clear, so the method is assumed to be the thin layer identification method of the sample, 15 batches of sample tests prove that the spots of the test sample are clear, and a negative reference sample is not interfered, so the method is assumed to be the identification item of the sample. The test methods and results are as follows:
the test method comprises the following steps: test by thin layer chromatography (rule 0502 of four parts of Chinese pharmacopoeia 2020 edition)
Sample solution preparation: taking 2g of the product, adding 50ml of methanol, carrying out ultrasonic treatment for 30 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 reference substance solution: and dissolving the protocatechuic acid reference substance with methanol to obtain reference substance solution with concentration of 1 mg/mL.
Thin layer chromatography conditions: thin layer plate: silica gel G thin layer plate; sample application amount: the sample solution and the reference substance solution are 1uL; developing agent: chloroform-ethyl acetate-methanol-formic acid (12:2:1:0.8); color-developing agent: spraying 2% ferric trichloride solution-1% potassium ferricyanide solution (1:1) (prepared by clinical use), and inspecting under sunlight.
Results: spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram. In the embodiment shown in FIG. 1, 15 batches of standard decoction TLC patterns are shown.
4. Determination of extract
The results of hot dipping method under the condition of 15 batches of standard decoction and ethanol as solvent according to the alcohol-soluble extract assay (general rule 2201 of Chinese pharmacopoeia 2020 edition) are shown in Table 2.
Table 2: extract measurement results
The result shows that the average value of 15 batches of standard decoction extract is 52.46%, the lower limit of the allowable range (average value 70% -130%) of the standard limit is referred, the alcohol-soluble extract of the product is not less than 37.0%, and the measurement results of 15 batches of standard decoction extract meet the requirement of the planned limit.
5. Feature profile testing
5.1 liquid chromatography
Chromatographic conditions: chromatographic column: c18 (250 mmx4.6mm,5 um) (Waters XSelec HSS T3); mobile phase: gradient elution was performed as specified in table 3 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 1.0mL/min; column temperature: 35 ℃; detection wavelength: 254nm.
Table 3:
(1) Preparing a reference solution: decocting rhizoma Cibotii reference material 1.0g in 25mL water for 1 hr, filtering, evaporating filtrate to dryness, adding methanol 5mL into residue, filtering, and collecting filtrate as reference solution;
(2) Preparing a reference substance solution: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol for dissolving, and preparing a reference substance solution with the concentration of 0.1 mg/mL;
(3) Preparing a test solution: taking 1.0g of a standard decoction sample of the scalded rhizoma cibotii, precisely weighing, placing the standard decoction sample into a conical bottle with a plug, adding 25mL of precisely weighed methanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking filtrate as a sample solution.
Assay: precisely sucking 10 μl of each of the reference solution, the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
5.2 methodology investigation
Investigation of the extraction method: respectively preparing test solution by different extraction methods, including ultrasonic 30min extraction and reflux 30min extraction, and measuring according to the test method 5.1. As shown in FIG. 2 and Table 4, the number of main peaks of ultrasonic wave for 30min is consistent with that of main peaks of reflux for 30min, and the total peak area RSD of the main peaks is 0.29%, so that the sample extraction mode is selected to be safer and more convenient ultrasonic treatment for 30 min.
Table 4:
investigation of extraction solvent: the test solutions were prepared with different extraction solvents, and were measured according to the test method of 5.1. As shown in Table 5, the results show that the number of main peaks is the same as that of the main peaks shown in FIG. 3, and the total peak area of the main peaks is the largest when the extraction solvent is methanol, so that the methanol is determined as the extraction solvent.
Table 5:
sample taking amount investigation: sample solutions with different sample amounts are prepared and measured according to a 5.1 test method. As shown in FIG. 4, the result showed that the sample amount was preferably 1.0g, so that the sample amount was determined to be 1.0g (see Table 6 for details).
Table 6: investigation of the results of the different sample taking amounts
Investigation of extraction time: sample solutions were prepared at different times for ultrasonic extraction, and were measured according to the test method 5.1 described above. As shown in FIG. 5, the difference of different extraction times is not large, the RSD is 1.42%, and the total area of main peaks of ultrasonic waves for 30 minutes is slightly large (see Table 7 in detail), so that the extraction time of a test sample is determined to be ultrasonic waves for 30 minutes.
Table 7: comparing and examining results of different extraction times
In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking about 1.0g of a standard decoction sample of the scalded rhizoma Cibotii, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of methanol, sealing, weighing, performing ultrasonic treatment for 20min, cooling, weighing again, supplementing the weight loss with methanol, shaking, and filtering.
5.3 feature map analysis method verification
Specificity investigation: the test sample was measured with 10. Mu.L of methanol as a solvent under 5.1 chromatography conditions, and the test showed that the blank solvent was free of interference as shown in FIG. 6.
Repeatability test: about 1.0g of the same batch of samples (batch number T210701) is taken, 6 parts are measured according to the condition of 5.1 chromatography, and the result shows that 6 peaks are shared in the characteristic spectrum of 6 samples to be tested, and the relative retention time RSD is less than 3%, so that the method has good reproducibility (see figure 7, table 8 and table 9 for details).
Table 8: repetitive test of characteristic pattern relative peak area
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD％ |
| S1 | 0.205 | 0.209 | 0.208 | 0.206 | 0.205 | 0.205 | 0.849 |
| S2 | 0.125 | 0.126 | 0.127 | 0.126 | 0.125 | 0.125 | 0.650 |
| S3 | 3.447 | 3.470 | 3.491 | 3.414 | 3.427 | 3.412 | 0.928 |
| S4(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| S5 | 0.928 | 0.928 | 0.928 | 0.928 | 0.928 | 0.928 | 0.000 |
| S6 | 0.083 | 0.084 | 0.083 | 0.083 | 0.082 | 0.082 | 0.909 |
Table 9: relative retention time of characteristic patterns for repeatability test
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD％ |
| S1 | 0.531 | 0.531 | 0.531 | 0.532 | 0.532 | 0.532 | 0.103 |
| S2 | 0.708 | 0.708 | 0.708 | 0.710 | 0.710 | 0.710 | 0.155 |
| S3 | 0.777 | 0.777 | 0.778 | 0.778 | 0.778 | 0.778 | 0.066 |
| S4(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| S5 | 1.106 | 1.106 | 1.107 | 1.107 | 1.107 | 1.107 | 0.047 |
| S6 | 1.465 | 1.465 | 1.464 | 1.467 | 1.467 | 1.467 | 0.091 |
Precision test: taking about 1.0g of samples in the same batch, measuring according to 5.1 chromatographic conditions, continuously injecting 6 needles for measuring, basically keeping the peak shape and the peak number consistent, and adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), evaluating the similarity of the designated 6 common characteristic peaks, wherein the relative retention time RSD is less than 3 percent (see fig. 8, table 10 and table 11 for details), and showing that the instrument precision is good.
Table 10: relative peak area of characteristic spectrum for precision test
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD％ |
| S1 | 0.186 | 0.186 | 0.186 | 0.187 | 0.186 | 0.186 | 0.219 |
| S2 | 0.113 | 0.113 | 0.113 | 0.112 | 0.112 | 0.113 | 0.458 |
| S3 | 3.102 | 3.101 | 3.100 | 3.102 | 3.096 | 3.094 | 0.109 |
| S4(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| S5 | 0.117 | 0.117 | 0.117 | 0.119 | 0.117 | 0.118 | 0.712 |
| S6 | 0.077 | 0.077 | 0.077 | 0.077 | 0.077 | 0.077 | 0.000 |
Table 11: relative retention time of precision test characteristic spectrum
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD％ |
| S1 | 0.532 | 0.532 | 0.532 | 0.532 | 0.532 | 0.531 | 0.077 |
| S2 | 0.710 | 0.710 | 0.710 | 0.709 | 0.709 | 0.709 | 0.077 |
| S3 | 0.778 | 0.779 | 0.778 | 0.778 | 0.778 | 0.778 | 0.052 |
| S4(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| S5 | 1.107 | 1.107 | 1.107 | 1.107 | 1.107 | 1.107 | 0.000 |
| S6 | 1.467 | 1.467 | 1.467 | 1.467 | 1.467 | 1.467 | 0.000 |
Stability test: taking about 1.0g of samples in the same batch, and carrying out measurement according to 5.1 chromatographic conditions, and respectively carrying out sample injection measurement at 0h, 2h, 4h, 8h, 12h and 24h, wherein the peak shape and the peak number of the characteristic spectrum are basically stable, and the relative retention time RSD is less than 3%, so that the sample solution is relatively stable within 24 hours. (see Table 12, table 13 and FIG. 9 for details) showing that the test sample solutions were stable for 24 hours.
Table 12: stability test characteristic spectrum relative peak area
| Peak number | 0 | 2h | 4h | 8h | 12h | 24h | RSD％ |
| S1 | 0.187 | 0.187 | 0.186 | 0.187 | 0.185 | 0.186 | 0.438 |
| S2 | 0.112 | 0.113 | 0.114 | 0.113 | 0.113 | 0.112 | 0.667 |
| S3 | 3.091 | 3.105 | 3.130 | 3.122 | 3.093 | 3.098 | 0.517 |
| S4(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| S5 | 0.103 | 0.103 | 0.104 | 0.102 | 0.103 | 0.102 | 0.732 |
| S6 | 0.077 | 0.077 | 0.077 | 0.077 | 0.077 | 0.077 | 0.000 |
Table 13: stability test characteristic pattern relative retention time
| Peak number | 0 | 2h | 4h | 8h | 12h | 24h | RSD％ |
| S1 | 0.532 | 0.531 | 0.531 | 0.531 | 0.532 | 0.531 | 0.097 |
| S2 | 0.709 | 0.709 | 0.708 | 0.708 | 0.709 | 0.709 | 0.073 |
| S3 | 0.778 | 0.777 | 0.777 | 0.777 | 0.777 | 0.778 | 0.066 |
| S4 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| S5 | 1.107 | 1.106 | 1.106 | 1.107 | 1.106 | 1.107 | 0.050 |
| S6 | 1.467 | 1.466 | 1.465 | 1.465 | 1.466 | 1.467 | 0.061 |
5.4 characterization analysis of Standard decoction feature atlas
Standard decoction characteristic spectrum measurement
According to the characteristic spectrum analysis method drawn by 5.3, 15 batches of scalding rhizoma cibotii decoction piece standard decoction and 15 batches of medicinal materials characteristic spectrums used for preparing the same are measured, and the result shows that 6 common peaks exist in the standard decoction and the traditional Chinese medicine decoction piece characteristic spectrums used for preparing the same and correspond to the retention time of 6 characteristic peaks in a reference substance chromatograph of a reference medicinal material, wherein the peak corresponding to a protocatechuic acid reference substance solution is peak 6, and the common peak characteristic spectrums are shown in figures 10 to 14 in detail.
Feature chromatogram similarity evaluation
The similarity evaluation system (2012 edition) of the traditional Chinese medicine chromatographic fingerprint image is adopted to evaluate the similarity of the selected 6 common characteristic peaks, and the result shows that the similarity of the characteristic chromatograms of the 15 batches of the standard decoction pieces of the scalding rhizoma cibotii is above 0.9, which indicates that the quality of the standard decoction is relatively stable. The relative retention time of the common peak and the S peak was calculated using the peak (4) corresponding to the protocatechuic acid reference peak as the S peak, and the relative retention time and the range thereof are shown in table 14.
Table 14: peak relative retention time of 15 batches of standard decoction
In summary, the standard decoction feature spectrum measurement method established by adopting the high performance liquid chromatography is adopted, and the established method is verified for precision, repeatability and stability according to the verification guiding principle (general rule 9101) of the four parts of analysis method in the edition 2020 of Chinese pharmacopoeia, so that the method meets the requirements. The 6 common characteristic peaks were calibrated, with peak 6 being protocatechuic acid. Calculating the relative retention time of another 5 characteristic peaks by taking the peak corresponding to the protocatechuic acid reference as an S peak, and respectively setting the average value of the relative retention time of 15 batches of sample peaks as a specified value to be: 0.53 (Peak 1), 0.71 (Peak 2), 0.78 (Peak 3), 1.11 (Peak 5), 1.47 (Peak 6), and the relative retention time allowable range was set to.+ -. 10% taking into consideration multi-factor errors of test operation, instrument, reagent, etc.
6. Content determination
6.1 test method
According to the content detection item of the scalding rhizoma cibotii in pharmacopoeia, protocatechuic acid is used as a content index component.
Test methods such as liquid chromatography in the above-described profile test.
The chromatographic conditions are as follows: c18 (250 mmx4.6mm,5 um) (Waters XSelec HSS T3); mobile phase: gradient elution was performed as specified in table 15 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 1.0ml per minute; column temperature: 35 ℃; detection wavelength: 254nm.
Table 15:
preparing a reference substance solution: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol to prepare a solution with the concentration of protocatechuic acid of 0.1mg/ml as the reference substance solution.
Preparing a test solution: taking about 1.0g of a standard decoction sample of the scalded rhizoma cibotii, precisely weighing, placing the standard decoction sample into a conical flask with a plug, precisely adding 25mL of methanol, sealing, weighing by ultrasonic for 30min, cooling, weighing again, supplementing the weight loss by using the methanol, shaking uniformly, and filtering to obtain a sample solution.
6.2 methodology investigation
Investigation of the extraction method: sample solutions are prepared by sample reflux and ultrasonic extraction methods, and the test is carried out according to the test method 6.1. The results showed that the sample was refluxed for 30min and was not very different from the ultrasound for 30min (power 250W, frequency 25 KHZ) and the RSD was 0.80%. (see Table 16 for details) the sample was chosen for more convenient and safe extraction by sonication for 30 minutes.
Table 16: comparison of protocatechuic acid content in different extraction methods
Investigation of extraction time: sample solutions were prepared at different extraction times, and were measured according to the test method 6.1 described above. The results show that the ultrasonic waves of the sample for 20min have little difference from the ultrasonic waves for 30min and 40min (the power is 250W, the frequency is 25 KHZ), the ultrasonic waves for 30min are slightly high, and the RSD is 0.76% (see Table 17 for details), so that the sample is selected more conveniently and safely by ultrasonic treatment for 30 min.
Table 17: comparison of protocatechuic acid content at different extraction times
Investigation of extraction solvent: sample solutions were prepared with different extraction solvents, and the measurement was performed according to the test method 6.1 described above. The results showed that the protocatechuic acid content was the highest and RSD was 0.76% when the extraction solvent was methanol (see table 18 for details), so methanol was determined as the extraction solvent.
Table 18: comparison of protocatechuic acid content of different extraction solvents
Sample size investigation: sample solutions were prepared in different amounts, and the measurement was performed according to the test method 6.1 described above. As a result, the sample amount of 1.0g was determined to be the optimum sample amount since the sample amount of 1.0g was determined to have a relatively high protocatechuic acid content (see Table 19 for details).
Table 19: comparison of protocatechuic acid content of different sample amounts
6.2 methodological validation of content determination
Repeatability test: about 1.0g of standard decoction samples of the same batch are taken, 6 parts of standard decoction samples are measured according to the test method of 6.1, the average value of the protocatechuic acid content in the samples is calculated to be 3.72mg/g, the RSD value is 054%, and the test shows that the method has good reproducibility (see Table 20 for details).
Table 20:
precision test: taking the protocatechuic acid reference substance solution shown in 6.1, continuously injecting 6 needles, measuring the peak area according to the test method of 9.1, and calculating the RSD value of the protocatechuic acid peak area to be 0.89%, which shows that the instrument precision is good (see Table 21 for details).
Table 21:
stability test: a batch of standard decoction samples of about 1.0g are taken, and are respectively sampled at 0h, 3h, 6h, 9h, 12h and 24h according to the test method of 9.1, the peak area is measured, the RSD value of the peak area is calculated to be 0.33%, and the test shows that the test sample solution is stable within 24 hours (see Table 22 for details).
Table 22:
linear range test: the protocatechuic acid reference solution (concentration of 0.105 mg/ml) was taken at 1ul, 5ul, 10ul, 15ul, 20ul, 25ul, respectively. The measurement was performed under the chromatographic conditions under 6.1. Drawing a standard curve by taking the area of the protocatechuic acid peak as an ordinate and the sample injection amount as an abscissa, and performing linear regression, wherein the regression equation is as follows: y=3e+06x+34842, r 2 The ratio of protocatechuic acid to peak area was found to have a good linear relationship in the range of 0.2625mg to 6.5625mg (see table 23, fig. 15).
Table 23: examination of the Linear relation of protocatechuic acid
Sample recovery rate test: about 0.5g of the sample (protocatechuic acid content: 3.72 mg/g) was precisely weighed, 6 parts in total, 10ml of a known concentration of a protocatechuic acid control solution (0.105 mg/ml) was added to each of the 6 parts of the sample, a sample solution was prepared by the method under 6.1, and the measurement was carried out under the chromatographic conditions under 6.1, whereby the average recovery rate of protocatechuic acid was 94.08% and the RSD was 0.73% (see Table 24 for details).
Table 24: sample recovery rate test results of protocatechuic acid
6.3 measuring the content of the traditional Chinese medicinal materials in the standard decoction
The raw material of the scalded rhizoma cibotii is initially processed into pieces with the length of about 1cm by a production place, and is processed into the scalded rhizoma cibotii decoction pieces after cleaning, and the raw catechin content of the scalded rhizoma cibotii decoction pieces is not changed only by screening without washing and baking in the cleaning process, so that the characteristic chromatogram of the scalded rhizoma cibotii decoction pieces and the raw catechin content refer to the medicinal material data.
According to the proposed content analysis method, 15 batches of the standard decoction of the scalded rhizoma cibotii and 15 batches of the protocatechuic acid content of the traditional Chinese medicine decoction pieces used for preparing the same are measured, and the results are shown in tables 25 and 26 in detail.
Table 25:15 batches of measurement results of protocatechuic acid of rhizoma cibotii traditional Chinese medicine decoction pieces
Table 26:15 batches of measurement results of protocatechuic acid in standard decoction of scalding rhizoma Cibotii
Protocatechuic acid content transfer rate: according to the detection method determined by standard decoction methodology research, the measurement results of 15 batches of standard decoction and the traditional Chinese medicine decoction pieces prepared and used by the standard decoction are calculated, the protocatechuic acid content transfer rate is mastered, the mass transfer condition is mastered, and a basis is provided for formulating the material internal control standard and the allowable range of characterization parameters of the material internal control standard. The standard decoction of rhizoma Cibotii is prepared by decocting rhizoma Cibotii decoction pieces with water for 2 times, concentrating the filtrate, and freeze drying. The transfer rate of protocatechuic acid content is shown in Table 27.
Table 27:15 batches of scalding rhizoma Cibotii standard decoction protocatechuic acid content transfer rate
From the data, the standard decoction of the scalded rhizoma cibotii is prepared by decocting the decoction pieces of the scalded rhizoma cibotii according to a scheme, the average content of protocatechuic acid in the decoction pieces of the scalded rhizoma cibotii is 1.92mg/g, and the measured content value range is 1.10-4.00mg/g, SD:1.12. the allowable range of the protocatechuic acid content is 1.35-2.50mg/g calculated according to 70% -130% of the average value, and the allowable range of the protocatechuic acid content of the standard decoction is 0.80-5.28 mg/g calculated according to the low limit of-SD and the high limit of +3SD.
From the data, the standard decoction of the scalded rhizoma cibotii decoction pieces is prepared by decocting the scalded rhizoma cibotii decoction pieces according to a scheme, the average transfer rate of protocatechuic acid is 64.02%, the measured value range of the transfer rate is 37.8-87.8%, and SD:19.43 according to the technical requirements of quality control and standard formulation of traditional Chinese medicine prescription granule, calculated according to 70-130% of average value, the allowable range of the content transfer rate of protocatechuic acid is 44.81-83.23%. The allowable range of the protocatechuic acid content transfer rate is 25.16 to 102.88 percent according to +/-2 SD. The result shows that the protocatechuic acid content and the transfer rate of 15 batches of standard decoction are within the allowable range, and can provide reference basis for the quality research of the scalding rhizoma cibotii formula particles.
According to the quality detection method for the standard decoction of the hot rhizoma cibotii, the quality of the standard decoction of the hot rhizoma cibotii is assessed through research on the properties of the standard decoction of the hot rhizoma cibotii, the dry extract extraction rate, thin-layer identification, extract, characteristic spectrum and protocatechuic acid content measurement and through multi-aspect measurement, a solid foundation is laid for the stable quality of products, a feasible quality standard of the decoction of the hot rhizoma cibotii can be established, effective control of the quality of the standard decoction of the hot rhizoma cibotii is realized, and a liquid phase analysis is carried out by adopting chromatographic conditions of the application, so that a chromatogram with better and clearer separation degree can be obtained. The standard decoction of the scalding rhizoma cibotii decoction pieces is prepared by a decoction method, the average content of protocatechuic acid is 1.92mg/g, the measured content range is 1.10-4.00mg/g, the SD (standard deviation) is 1.12, the allowable range of protocatechuic acid content is 1.35-2.50mg/g according to the average value of 70-130%, and the allowable range of protocatechuic acid content of the standard decoction is 0.80-5.28 mg/g according to the low limit of-SD and the high limit of-3 SD. The average transfer rate of protocatechuic acid is 64.02%, the transfer rate range is 37.8% -87.8%, SD is 19.43, according to the technical requirements of quality control and Standard establishment of traditional Chinese medicine prescription granule, the allowable range of protocatechuic acid content transfer rate is calculated according to 70% -130% of the transfer rate average value, and the allowable range of protocatechuic acid content transfer rate is 44.81-83.23%. The allowable range of the protocatechuic acid content transfer rate is 25.16 to 102.88 percent according to +/-2 SD. According to the data, the limit of measuring the content of protocatechuic acid in the standard decoction is calculated as follows: 0.80 mg/g-5.20 mg/g, and the allowable range of the content transfer rate is 25.0% -100.0%. The results show that the protocatechuic acid content and the transfer rate of the protocatechuic acid in the standard decoction of a plurality of batches are all within the allowable range, so the application can provide reference for the quality standard research of the scalded rhizoma cibotii formula particles.
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 application (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the application, the steps may be implemented in any order and there are many other variations of the different aspects of the application as described above, which are not provided in detail for the sake of brevity.
The embodiments of the application are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present application should be included in the scope of the present application.
Claims (3)
1. A method for detecting the quality of a standard decoction of a hot rhizoma cibotii is characterized by comprising the following detection methods,
the standard decoction content standard is limited to 0.80-5.20mg of protocatechuic acid per 1g by measuring the characters of the standard decoction of the scalded rhizoma Cibotii, the extract yield of the dry extract, the thin-layer identification, the extract, the characteristic map and the protocatechuic acid content, wherein the extract yield of the dry extract is measured by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the protocatechuic acid content are measured by liquid chromatography;
the determination of the characteristic spectrum by liquid chromatography comprises: analyzing with liquid chromatograph, taking the solution prepared by scalding rhizoma Cibotii reference medicine as reference substance solution, the solution prepared by protocatechuic acid reference substance as reference substance solution, and the solution prepared by scalding rhizoma Cibotii standard decoction sample as test substance solution, respectively precisely sucking the reference substance solution, the reference substance solution and the test substance solution, respectively injecting into liquid chromatograph, and measuring to obtain the final product; wherein, the chromatographic condition adopted is C18, the column length is 250mm, the column inner diameter is 4.6mm, and the grain diameter is 5um,Waters XSelec HSS T3; mobile phase: using methanol as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, and performing gradient elution according to the specification of a table a;
table a gradient elution procedure
Flow rate: 1.0mL/min; column temperature: 35 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm;
the thin layer chromatography comprises the following steps:
s11: preparing a test sample solution a: taking 2g of a standard decoction sample of the scalded rhizoma Cibotii, adding 50mL of methanol, carrying out ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, and dissolving residues with 1mL of methanol to prepare a sample solution a;
s12: preparing a reference substance solution a: dissolving protocatechuic acid reference substance in methanol to obtain reference substance solution a with concentration of 1 mg/mL;
s13: thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel G thin layer plate; sample application amount: 1uL of each of the sample solution a and the reference solution a; developing agent: chloroform-ethyl acetate-methanol-formic acid with a volume ratio of 12:2:1:0.8; color-developing agent: temporarily preparing 2% ferric trichloride solution-1% potassium ferricyanide solution, wherein the volume ratio of the 2% ferric trichloride solution to the 1% potassium ferricyanide solution is 1:1, and inspecting under sunlight;
the characteristic spectrum determination by liquid chromatography further comprises the following steps:
s21: preparation of reference solution b: decocting rhizoma Cibotii control 1.0g in 25mL water for 1 hr, filtering, evaporating filtrate to dryness, adding 5mL methanol into the residue, filtering, and collecting filtrate as reference solution b;
s22: preparing a reference substance solution b: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol for dissolving, and preparing a reference substance solution b with the concentration of 0.1 mg/mL;
s23: preparing a test sample solution b: taking 1.0g of a standard decoction sample of the scalded rhizoma cibotii, precisely weighing, placing the standard decoction sample into a conical bottle with a plug, adding 25mL of precisely weighed methanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking filtrate as a sample solution b;
the determination of protocatechuic acid content by liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from protocatechuic acid reference substance as a reference substance solution c, taking a solution prepared from a standard decoction sample of the hot rhizoma Cibotii as a sample solution c, precisely sucking the reference substance solution c and the sample solution c respectively, respectively injecting into the liquid chromatograph, and measuring to obtain the final product; wherein, the chromatographic condition adopted is C18, the column length is 250mm, the column inner diameter is 4.6mm, and the grain diameter is 5um,Waters XSelec HSS T3; mobile phase: methanol 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 a specified rule; flow rate: 1.0mL/min; column temperature: 35 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm;
the method for measuring the content of protocatechuic acid by adopting the liquid chromatography further comprises the following steps:
s31: preparing a reference substance solution: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol to prepare a solution with the concentration of the protocatechuic acid of 0.1mg/ml, and taking the solution as reference substance solution c;
s32: preparing a test solution: taking 1.0g of a standard decoction sample of the scalded rhizoma cibotii, precisely weighing, placing the standard decoction sample into a conical flask with a plug, precisely adding 25mL of methanol, sealing, weighing by ultrasound for 30min, cooling, weighing again, supplementing the weight loss by using the methanol, shaking uniformly, and filtering to obtain a sample solution c.
2. The method for detecting the quality of a standard decoction of a hot rhizoma Cibotii according to claim 1, wherein the decoction method comprises: soaking rhizoma Cibotii decoction pieces in water for 30-40min, decocting twice for 30-40min for the first time and 25-30min for the second time, separating solid from liquid while hot, mixing filtrates, concentrating, and drying to obtain rhizoma Cibotii standard decoction dry extract powder.
3. The method for detecting the quality of a standard decoction of a hot-stamped cibotium barometric pressure as claimed in claim 1, wherein the hot-dip method uses ethanol as a solvent, and the range of the extract is measured by a hot-dip method under the alcohol-soluble extract measurement method.
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