CN113848278A - Quality control method for standard decoction of radix Cudraniae - Google Patents
Quality control method for standard decoction of radix Cudraniae Download PDFInfo
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Abstract
The invention provides a quality control method of a radix cudramiae standard decoction, which is characterized in that the content range of the taxifolin of the standard decoction is drawn up to be 5.0-83.0mg/g through the characteristics, the dry extract extraction rate, the thin layer identification, the extract, the characteristic map and the taxifolin content measurement of the radix cudramiae standard decoction; wherein, the extraction rate of the dry extract is measured by adopting a decoction method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the taxifolin content are determined by liquid chromatography. The quality of the standard decoction of the radix cudraniae can be effectively controlled by researching the characters of the standard decoction of the radix cudraniae, the extraction rate of dry extract, thin-layer identification, extract, characteristic spectrum and the content measurement of the taxifolin.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality control method of a cudrania cochinchinensis standard decoction.
Background
Radix Cudraniae is fresh or dried root of fructus Broussonetiae. Collected all the year round, the rootlets are cut off, cleaned, used fresh or cut off, sliced and dried in the sun. According to the records of Chuanchuanshi, Bencao Shiyi, Kuo Gen (the prescription of Qianjin for emergency), Chuansha (the preparation of raw herb for medicinal property), Digossypii radix, Laiu Daishi Shi (the record of Lingnan vegetable medicine), 33896, and Zhi (Hunan). Is root of Cudrania tricuspidata or Brookra of Moraceae. Chuan Shi is named in Ling nan Cai Yao Bian (Ling nan Cai Yao Bian). The book "herbal shiyi" carries with "nu cudrania", which means: "Shengjiangnan mountain field. Cudrania tricuspidata, spiny thorn, irregular winter. "compendium of materia Medica": "the tree is small like a Chinese thorny. The leaves are also as small as tussah leaves and can be used for feeding silkworm, the above description is consistent with the case of brothera. The compendium of materia Medica also carries "Cudrania", cloud: "everywhere there is a mountain, which is like fasciculation, dry, loose and straight. The leaves are thick and thick, rounded and sharp. 'paper mulberry, Jiu-layer peel, false litchi, monkey joy, mountain litchi, gold thorn, bird's dead, rat thorn, rice ball , wild plum fruit. Collected all the year round, the rootlets are cut off, cleaned, used fresh or cut off, sliced and dried in the sun.
The radix Cudraniae medicinal material contains flavonoids, phenols, organic acids, etc., and the radix Cudraniae medicinal material has effects of invigorating spleen, benefiting stomach, relieving rigidity of muscles, activating collaterals, dispelling pathogenic wind, removing dampness, and removing blood stasis; it is indicated for lumbago, arthralgia, consumptive disease, yellow swelling, spleen deficiency and diarrhea. At present, no relevant standard of the drug of radix cudraniae has been established. The prior art only relates to the inspection of the properties of the drug of the cudrania cochinchinensis, has weak pertinence, and the purpose of really controlling the quality of the drug is difficult to achieve by using the methods.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a quality control method of a standard decoction of radix cudraniae, aiming at conveniently and effectively evaluating and controlling the internal quality of the radix cudraniae medicinal material.
Based on the above purposes, the invention provides a quality control method of a radix cudramiae standard decoction, which is characterized in that the content range of the taxifolin of the standard decoction is drawn up to be 5.0-83.0mg/g by determining the characters, the dry extract yield, the thin layer identification, the extract, the characteristic map and the taxifolin content of the radix cudramiae standard decoction; wherein, the extraction rate of the dry extract is measured by adopting a decoction method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the taxifolin content are determined by liquid chromatography.
The limiting range of the dry extract yield is 4.0-7.5%.
The thin layer chromatography comprises the following steps:
a1, adding methanol into the radix Cudraniae decoction, performing ultrasonic treatment, filtering, evaporating the filtrate, and dissolving the residue in methanol to obtain a sample solution a;
a2, decocting radix Cudraniae control material, filtering, evaporating filtrate, and dissolving residue in methanol to obtain control solution a;
a3, setting thin-layer chromatography conditions: silica gel G thin layer plate; sample amount of spotting: the solution a of the test sample is 5ul, and the solution a of the reference medicinal material is 10 ul; developing agent: mixing dichloromethane, ethyl acetate and methanol in a volume ratio of 12:5: 2; and (5) placing under an ultraviolet lamp for inspection.
The hot dipping method uses ethanol as solvent, and the extract content measured by hot dipping method in alcohol-soluble extract measuring method is not less than 46.5%.
The characteristic spectrum and the taxifolin content are determined by liquid chromatography, which comprises respectively sucking the reference solution b and the sample solution b of radix Cudraniae and injecting into a liquid chromatograph for determination; wherein, the adopted chromatographic conditions are as follows: a chromatographic column: c18; mobile phase: performing gradient elution by using methanol as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; flow rate: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 290 nm.
The test solution b is prepared by collecting radix Cudraniae standard decoction sample, adding 25 times of 70% methanol, sealing, subjecting to ultrasound treatment, cooling, weighing, supplementing with 70% methanol, reducing weight loss, shaking, and filtering.
The method for measuring the characteristic spectrum by adopting the liquid chromatography further comprises the steps of injecting the test samples of the same batch into the liquid chromatograph, measuring and evaluating the similarity of the established common characteristic peaks.
The method for measuring the characteristic map and the content of the taxifolin by adopting the liquid chromatography further comprises the steps of feeding samples of the test samples of the same batch for 0h, 3h, 6h, 9h, 12h and 24h respectively, measuring the peak shape and the peak number, and evaluating the similarity of the common characteristic peaks for testing the stability of the test sample solution.
The method for measuring the characteristic spectrum and the content of the taxifolin by adopting the liquid chromatography further comprises the steps of continuously feeding 6 needles to the test samples in the same batch to measure the peak shape and the peak number, and evaluating the similarity of the common characteristic peaks for testing whether the precision of a chromatograph is good or not.
The method for measuring the content of the taxifolin by adopting the liquid chromatography also comprises the step of weighing a test sample, adding the test sample into a taxifolin reference solution, and testing the average sample adding recovery rate of the taxifolin.
The invention has the beneficial effects that:
1. according to the method, the properties of the standard decoction of the radix cudraniae, the dry extract yield, the thin-layer identification, the extract, the characteristic spectrum and the content determination of the taxifolin are researched, so that the quality of the standard decoction of the radix cudraniae can be effectively controlled.
2. The radix cudramiae standard decoction is prepared by carrying out ultrasonic extraction on radix cudramiae, the average content of taxifolin is 29.08mg/g, the content range is 4.97-54.18 mg/g, and the SD is 18.01; calculated according to the mean value +/-3 SD, the allowable range of the content of the taxifolin is-24.95-83.11 mg/g. Since-3 SD is a negative value, the lowest value of the measured values, 4.97, is the lower limit. Therefore, the total content range of the taxifolin in the standard decoction is drawn as follows: 5.0 to 83.0 mg/g. The result shows that the total content of the taxifolin in the standard decoction of the cudrania cochinchinensis and the transfer rate thereof are both in an allowable range, and can provide reference basis for the quality research of formula granules of the cudrania cochinchinensis (spina gleditsiae).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a TLC pattern of a 15 lot standard decoction of the present invention; wherein, A, negative control; s, comparing the medicinal materials; 1-15, a test article;
FIG. 2 is a comparison of different extraction methods;
FIG. 3 is a comparison graph of different extraction times;
FIG. 4 is a comparison of different extraction solvents;
FIG. 5 is a graph comparing different solvent amounts;
FIG. 6 is a comparison of blank solvents;
FIG. 7 is a common peak superposition signature for a reproducibility test;
FIG. 8 shows the precision measurement common peak overlap profile;
FIG. 9 is a stability test consensus peak overlay signature;
FIG. 10 is a taxifolin reference map;
FIG. 11 is a graph of a control drug of radix Cudraniae;
FIG. 12 is a graph of the line survey of taxifolin.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined. The use of "first," "second," and similar terms in this disclosure is not intended to indicate any order, quantity, or importance, but rather is used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", and the like are used merely to indicate relative positional relationships, and when the absolute position of the object being described is changed, the relative positional relationships may also be changed accordingly.
The invention provides a quality control method of a radix cudramiae standard decoction, which is characterized in that the content range of the taxifolin of the standard decoction is drawn up to be 5.0-83.0mg/g through the characteristics, the dry extract extraction rate, the thin layer identification, the extract, the characteristic map and the taxifolin content measurement of the radix cudramiae standard decoction; wherein, the extraction rate of the dry extract is measured by adopting a decoction method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the taxifolin content are determined by liquid chromatography. The following is a description by specific examples.
The preparation method comprises the following steps: referring to the management standard of traditional Chinese medicine decoction rooms of medical institutions (No. 2009 of the State administration of traditional Chinese medicine) relevant parameters such as a pretreatment method, decoction times, water addition amount, decoction time and the like are fixed for decoction, and then solid-liquid separation, concentration and drying are carried out.
Determination of cream yield
Taking 15 batches of radix cudraniae (radix spina gleditsiae) decoction pieces, preparing 15 batches of standard decoction dry extract powder according to the preparation method, calculating the dry extract yield (see table 1 below) by using the dry extract powder, calculating the average yield to be 5.68%, calculating according to the allowable range of standard decoction plaster yield (the average value is 70-130%), wherein the allowable range of plaster yield is 3.976-7.384%, and the allowable range of plaster yield of standard decoction prepared from radix cudraniae (radix spina gleditsiae) decoction pieces is 4.0-7.5%.
TABLE 1 percentage of standard decoction of Chuanshao stone (radix Broussonetiae)
The results show that the cream yield of 15 batches of standard decoction is 4.6-7.3%, and the standard decoction meets the set limit range of 4.0-7.5%.
The characteristics are as follows: according to the physical characteristics of 15 batches of standard decoction, the standard decoction is described as light yellow to earthy yellow powder; light smell, bland taste.
Thin-layer identification: the product is a dry extract of single-ingredient decoction piece cudrania cochinchinensis (radix spina), reference is made to a reference literature cudrania cochinchinensis thin-layer identification method, cudrania cochinchinensis (radix spina) is used as a reference medicinal material, the thin-layer identification method is established, and through 15 batches of sample tests, the spot of a test sample is clear, and a negative reference sample is free of interference, so that the product is drawn as an identification item. The test methods and results are as follows:
the test method comprises the following steps: performing thin layer chromatography (China pharmacopoeia 2020 edition four-part general rule 0502)
Preparing a test solution: taking about 1g of the product, adding 20ml of methanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues for dissolving to obtain the product.
Control solution: collecting control material of radix Cudraniae (fructus Hippophae) 5g, adding water 50ml, boiling for 30 min, filtering, evaporating filtrate to dryness, adding methanol 20ml into residue, and making into control material solution by the same method.
Thin-layer chromatography conditions: thin-layer plate: silica gel G thin layer plate; sample amount of spotting: sucking 5ul of test solution and 10 mul of reference solution; developing agent: dichloromethane-ethyl acetate-methanol (12:5: 2); and (6) inspection: inspecting under an ultraviolet lamp (365 nm).
As a result: in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution. The TLC pattern of the 15 batches of standard decoction is shown in FIG. 1.
And (3) extract determination: taking 15 batches of standard decoction, taking ethanol as solvent, and performing hot-dipping assay under alcohol-soluble extract assay (China pharmacopoeia 2020 Ed. 2201), and the results are shown in Table 2 below.
TABLE 2 extract measurement results
The results show that the average value of 15 batches of standard decoction extract is 66.41%, and the alcohol-soluble extract of the product is drawn to be not less than 46.5% by referring to the lower limit of the allowable range (average value is 70-130%) of the paste yield of the standard decoction. The measurement results of 15 batches of standard decoction all meet the requirement of a set limit.
Testing of feature maps
Determination of measurement wavelength: reference to a reference determines a detection wavelength of 290 nm.
Test method
Chromatographic conditions are as follows: a chromatographic column: c18(250mmx4.6mm, 5um) (Shim-pack GIST); mobile phase: gradient elution was performed with methanol as mobile phase a and 0.1% phosphoric acid as mobile phase B as specified in table 3 below; flow rate: 1.0ml per minute; column temperature: 30 ℃, detection wavelength: 290 nm.
TABLE 3
Preparation of reference solutions: taking 0.5g of a cudrania cochinchinensis (spina gleditsiae) reference medicinal material, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the weight loss by 70% methanol, shaking up, filtering, and taking a subsequent filtrate as a reference medicinal material solution. Taking appropriate amount of taxifolin control, and adding 70% methanol to obtain control solution containing 70ug per 1 ml.
Preparation of a test solution: taking about 0.1g of a cudrania cochinchinensis (radix Berchemiae Kadsurae) standard decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing weight loss with 70% methanol, shaking up, and filtering to obtain the final product.
The determination method comprises the following steps: precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.
Methodology investigation
Investigation of extraction method: preparing test solution by different extraction methods (ultrasonic and reflux), and determining according to the test method in the characteristic spectrum test. The results show that the two extraction methods have the same number of main peaks, and the peak shape and the separation degree are both in accordance with the regulations (as shown in figure 2), so that the sample extraction method is selected to be simple and convenient ultrasonic treatment for 30 minutes.
And (3) extracting time investigation: the test solutions were prepared at different sonication times (30 min, 45 min, 60 min) and measured as described above. The results show that the number of main peaks extracted in different extraction times is consistent, and the peak shape and the separation degree are both in accordance with the regulations (as shown in figure 3), so that 30 minutes of ultrasound with shorter extraction time is determined as the extraction time.
Investigation of extraction solvent: the test solutions were prepared with different extraction solvents (methanol, 70% ethanol) and tested according to the test methods described above. The results show that the extraction main peaks of different extraction solvents are consistent in number, but the 70% methanol peak shape and the separation degree are better (as shown in figure 4), so that the 70% methanol is determined to be used as the extraction solvent.
Sample sampling amount investigation: the test solutions were prepared in different amounts of solvent (15ml,25ml,50ml) and tested according to the test methods described above. The results show that the number of main extraction peaks is consistent with that of the main extraction peaks extracted by different solvent dosages, the peak shape and the separation degree are both in accordance with the regulations, but when the solvent dosage is 25ml, the sample content value is the largest (as shown in figure 5), and the solvent dosage is determined to be 25 ml.
In summary, the main parameters for determining the preparation method of the test solution are as follows: taking a standard decoction sample of cudrania cochinchinensis (spina n.) kudo of about 0.1g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% methanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing with 70% methanol to reduce weight loss, shaking up, and filtering to obtain the final product.
Verification of characteristic spectrum analysis method
And (3) special investigation: taking 10ul of 70% methanol alcohol as a solvent for a test sample, and determining according to the chromatographic conditions in the characteristic spectrum test. The test shows that: blank solvent was not interfered (as shown in figure 6).
And (3) repeatability test: taking about 0.1g and 6 parts of samples in the same batch, and determining according to a method in a characteristic spectrum test, wherein the results show that 4 common peaks exist in the characteristic spectrum of the 6 test samples, and performing similarity flattening on the specified 4 common characteristic peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), wherein the relative retention time RSD is less than 3 percent and the relative peak area RSD is less than 5 percent (see the following table 4 and table 5), which indicates that the method has good reproducibility.
TABLE 4 relative retention time of feature profile for reproducibility test
| | Repeatability | 1 | |
|
|
Repeatability 5 | |
RSD(％) |
| 1 | 0.445 | 0.445 | 0.446 | 0.447 | 0.447 | 0.447 | 0.220 | |
| 2 | 0.783 | 0.784 | 0.784 | 0.784 | 0.784 | 0.785 | 0.081 | |
| 3(S) | 1 | 1 | 1 | 1 | 1 | 1 | / | |
| 4 | 1.475 | 1.475 | 1.476 | 1.476 | 1.476 | 1.475 | 0.037 |
TABLE 5 relative peak area of characteristic spectrum for repeatability test
And (3) precision test: about 0.1g of samples in the same batch are taken, 6 needles are continuously injected for measurement according to the test method in the characteristic spectrum test, the peak shape and the peak number are basically consistent, a similarity flat price is carried out on 4 specified common characteristic peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the relative retention time RSD is less than 3 percent, and the relative peak area RSD is less than 5 percent (see the following tables 6 and 7), so that the instrument has good precision.
TABLE 6 relative retention time of the feature profile for the precision test
| | Precision | 1 | |
|
|
Precision 5 | |
RSD(％) |
| 1 | 0.445 | 0.446 | 0.445 | 0.445 | 0.445 | 0.446 | 0.116 | |
| 2 | 0.783 | 0.783 | 0.783 | 0.783 | 0.783 | 0.783 | 0.000 | |
| 3(S) | 1 | 1 | 1 | 1 | 1 | 1 | / | |
| 4 | 1.477 | 1.476 | 1.477 | 1.476 | 1.475 | 1.477 | 0.055 |
TABLE 7 relative peak area of characteristic spectrum for precision test
| | Precision | 1 | |
|
|
Precision 5 | |
RSD(％) |
| 1 | 1.330 | 1.333 | 1.333 | 1.336 | 1.337 | 1.339 | 0.259 | |
| 2 | 0.054 | 0.053 | 0.053 | 0.052 | 0.052 | 0.051 | 1.616 | |
| 3(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 | |
| 4 | 0.323 | 0.323 | 0.324 | 0.324 | 0.323 | 0.325 | 0.240 |
And (3) stability test: taking about 0.1g of a batch of samples, respectively injecting samples at 0h, 3h, 6h, 9h, 12h and 24h according to the test method in the characteristic spectrum test to determine, wherein the peak shape and the peak number are basically stable, and performing similarity evaluation on 4 specified common characteristic peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), wherein the relative retention time RSD is less than 3 percent and the relative peak area RSD is less than 5 percent (see the following table 8 and table 9), which indicates that the solution of the sample is stable within 24 hours. The stability test consensus peak overlap profile is shown in figure 7.
TABLE 8 stability test feature profiles relative retention time
| |
0 |
3 |
6 |
9 |
12 hours | 24 hours | RSD(％) |
| 1 | 0.445 | 0.445 | 0.446 | 0.446 | 0.447 | 0.447 | 0.201 |
| 2 | 0.783 | 0.783 | 0.783 | 0.784 | 0.784 | 0.784 | 0.070 |
| 3(S) | 1 | 1 | 1 | 1 | 1 | 1 | / |
| 4 | 1.477 | 1.476 | 1.477 | 1.476 | 1.477 | 1.475 | 0.055 |
TABLE 9 relative peak area of characteristic spectra for stability test
| |
0 |
3 |
6 |
9 |
12 hours | 24 hours | RSD(％) |
| 1 | 1.330 | 1.336 | 1.339 | 1.345 | 1.349 | 1.366 | 0.936 |
| 2 | 0.054 | 0.052 | 0.051 | 0.050 | 0.049 | 0.056 | 4.723 |
| 3(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | / |
| 4 | 0.323 | 0.324 | 0.325 | 0.313 | 0.314 | 0.331 | 2.142 |
Characterization and analysis of standard decoction characteristic spectrum
Determination of standard decoction characteristic map
According to the proposed characteristic spectrum analysis method, the characteristic spectrums of 15 batches of the standard decoction of the radix cudraniae (the radix spina serratae) and 15 batches of the Chinese herbal pieces prepared and used by the standard decoction of the radix cudraniae (the radix spina serratae) are determined, and the results show that the characteristic chromatogram of the standard decoction and the Chinese herbal pieces prepared and used by the standard decoction have 4 common peaks, and the common peaks correspond to the retention time of 4 characteristic peaks in the chromatogram of a reference substance of a reference medicinal material, wherein the peak corresponding to the reference substance of the taxifolin is peak 3, and the common peak characteristic spectrum (as shown in figure 8).
And (3) evaluating the similarity of the characteristic chromatograms: the similarity evaluation system (2012 edition) is adopted to evaluate the similarity of the selected 4 common characteristic peaks, and the result shows that the similarity of the characteristic chromatograms of the standard decoction of 15 batches of radix cudraniae (radix spina gleditsiae) decoction pieces is more than 0.9, which indicates that the quality of the standard decoction is relatively stable. The peak (3) corresponding to the taxifolin reference peak is taken as the S peak, and the relative retention time of the common peak and the S peak is calculated, and the relative retention time and the range are shown in the following table 10.
TABLE 1015 Standard decoction batches shared peak relative retention time
In conclusion, the method for determining the standard decoction characteristic spectrum established by the high performance liquid chromatography is adopted, and the established method is verified in terms of precision, repeatability and stability according to the analysis method verification guiding principle (general rule 9101) of the four parts of the Chinese pharmacopoeia 2020 edition, and meets the requirements. Similarity evaluation is carried out on the characteristic spectrums of 15 batches of standard decoction samples by adopting a traditional Chinese medicine chromatogram fingerprint image similarity evaluation system (2012 edition), 4 common characteristic peaks are calibrated, and the peak 3 is taxifolin. Taking the peak corresponding to the taxifolin reference substance as an S peak, calculating the relative retention time of other 3 characteristic peaks, and drawing up the average value of the relative retention time of the peaks of 15 batches of samples as specified values: 0.46 (peak 1), 0.78 (peak 2), 1.47 (peak 4), considering the error of experiment operation, instrument, reagent and other multifactor, the relative retention time allowed range is defined as + -10%.
Content determination: the chemical components of the radix cudramiae medicinal material are flavonoids, phenols, organic acids and the like, and the components such as taxifolin, quercetin, kaempferol and the like are detected in the radix cudramiae. Has effects in invigorating spleen, benefiting stomach, relaxing muscles and tendons, activating collateral flow, dispelling pathogenic wind, removing dampness, and removing blood stasis; it is indicated for lumbago, arthralgia, consumptive disease, yellow swelling, spleen deficiency and diarrhea. In addition, the effective component of the taxifolin in the cudrania root has certain protection effects of resisting cervical cancer, lung cancer and myocardial ischemia reperfusion injury. Therefore, the taxifolin is used as the content index component of the standard decoction of radix Cudraniae (spine) in the process.
Test method
Chromatographic conditions are as follows: a chromatographic column: c18(250mmx4.6mm, 5um) (Shim-pack GIST); mobile phase: using methanol as mobile phase A and 0.1% phosphoric acid as mobile phase B, and performing gradient elution according to the specification in the following table; flow rate: 1.0ml per minute; column temperature: 30 ℃, detection wavelength: 290 nm.
Preparation of control solutions: accurately weighing appropriate amount of taxifolin, adding 70% methanol to obtain reference solution containing 70 μ g of taxifolin per 1ml, and filtering.
Preparation of a test solution: taking about 0.1g of a cudrania cochinchinensis (radix Berchemiae Kadsurae) standard decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing weight loss with 70% methanol, shaking up, and filtering to obtain the final product.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Methodology investigation
Investigation of extraction method: preparing test solution by different extraction methods (ultrasonic and reflux), and determining according to the test method in the standard decoction characteristic spectrum determination. The result shows that the difference between the content of the taxifolin of the sample after refluxing for 30 minutes and the content of the taxifolin after ultrasonic treatment (power 250W and frequency 25KHZ) for 30 minutes is large, the RSD is 3.16 percent, and the ultrasonic treatment is adopted as the extraction mode for 30 minutes.
TABLE 11 comparison of different extraction methods
Examination of extraction time: the test solutions were prepared at different extraction times (30 min, 45 min, 60 min) and tested according to the test method 9.1 described above. The result shows that the difference of the content of the taxifolin of the sample in 30 minutes of ultrasonic treatment and 45 minutes of ultrasonic treatment and 60 minutes of ultrasonic treatment is not large, and the RSD is 1.35 percent. (see table 12 below), the sample treatment time was chosen to be 30 minutes of sonication.
TABLE 12 comparison of different extraction times
Investigation of extraction solvent: the test solutions were prepared with different extraction solvents (methanol, 70% ethanol) and tested according to the test methods described above. The results show that the content of the taxifolin in different extraction solvents is not obviously different, and the content of the taxifolin is the highest when the extraction solvent is 70 percent methanol (see the following table), so that the 70 percent methanol is determined as the extraction solvent.
TABLE 13 comparison of different extraction solvents
Investigation of solvent dosage: the test solutions were prepared in different amounts of solvent (15ml,25ml,50ml) and tested according to the test methods described above. The result shows that the content of the taxifolin is the highest when the dosage of the solvent is 25ml, so that the dosage of the solvent is determined to be 25 ml.
TABLE 14 comparison of different solvent amounts
Verification of content determination methodology
And (3) repeatability test: about 0.1g of standard decoction samples of the same batch are taken, 6 parts in total are measured according to the test method, the content average value of the samples is 23.6616mg/g, the RSD value is 0.40%, and the test shows that the method has good reproducibility (see the following table 15).
TABLE 15 repeatability tests
And (3) precision test: taking a sample solution adopted in the standard decoction characteristic spectrum determination, continuously injecting a sample of 6 needles, determining the peak area according to the test method, and calculating the RSD value of the peak area in the sample to be 0.21%, which indicates that the instrument precision is good (see the following table 16).
TABLE 16 precision test
And (3) stability test: about 0.1g of a batch of decoction samples are taken, sample injection is carried out for 0h, 3h, 6h, 9h, 12h and 24h respectively according to the test method in the standard decoction characteristic spectrum determination, the peak areas are determined, the RSD value of the peak areas is calculated to be 0.70%, and tests show that the solution of the test sample is stable within 24 hours (see the following table 17).
TABLE 17 stability test
Linear range test: taking the taxifolin reference solution (the concentration is 0.27593mg/ml), measuring 5ml to 10ml volumetric flasks with the concentration of 0.13796 mg/ml; same way, dilution by half, concentration: 0.06898 mg/ml; 0.03449 mg/ml; 0.01724 mg/ml; 0.00862 mg/ml. And (4) determining according to the chromatographic conditions in the standard decoction characteristic spectrum determination. Drawing a standard curve by taking the ordinate of the area of the taxifolin peak and the concentration as the abscissa, and performing linear regression, wherein the linear equation of the taxifolin is as follows: y is 35,859,910.7468x +27,041.3807, and R2 is 0.9999, so that the taxifolin is 0.00862 mg-0.27593 mg, and has a good linear relationship with the peak area (see table 18 and fig. 12 below).
TABLE 18 Linear test results for taxifolin
Sample recovery rate test: precisely weighing about 0.05g of a sample (the content of the taxifolin is 23.6616mg/g) and 6 parts in total, adding 1ml of a taxifolin reference substance solution (0.91977mg/ml) with known concentration respectively, preparing a sample solution according to a method in standard decoction characteristic spectrum measurement, measuring according to chromatographic conditions in the standard decoction characteristic spectrum measurement, and calculating that the average sample adding recovery rate of the taxifolin is 96.29 percent and the RSD is 1.42 percent.
TABLE 19 sample recovery test results
Measuring the contents of the standard decoction and the traditional Chinese medicinal materials: processing radix Cudraniae (fructus Broussonetiae) into pieces, cleaning, and processing into radix Cudraniae (fructus Broussonetiae) decoction pieces, wherein the radix Cudraniae (fructus Broussonetiae) decoction pieces are not washed with water and baked during cleaning process, and the content of taxifolin is not changed by screening; therefore, the characteristic chromatogram of the radix cudraniae (radix spina serratae) decoction pieces and the content of the taxifolin refer to the medicinal material data.
According to the proposed content analysis method, the contents of 15 batches of the standard decoction of radix Cudraniae (radix Broussonetiae) and 15 batches of the taxifolin prepared from the Chinese medicinal decoction pieces are determined, and the results are shown in the following table 20.
TABLE 2015 measurement of radix Cudraniae (fructus Broussonetiae) Chinese medicinal materials and radix Panacis Quinquefolii slice
Measurement result of 15 batches of radix Cudraniae (Broussonetia papyrifera) standard decoction taxifolin
Content transfer rate: according to the detection method determined by standard decoction methodology research, the content transfer rate of the taxifolin is calculated for 15 batches of standard decoction and the determination results of the prepared traditional Chinese medicine decoction pieces, the mass transfer condition of the taxifolin is mastered, and a basis is provided for formulating the internal control standard of the materials and the allowable range of the characterization parameters of the internal control standard. The standard decoction is prepared by decocting radix Cudraniae (fructus Broussonetiae) decoction pieces in water for 2 times, concentrating the filtrate, and freeze drying. The rate of transfer of the taxifolin content (see table 21 below).
TABLE 2115 Standard decoction of radix Cudraniae (Broussonetia papyrifera) for taxifolin content transfer rate
According to the data, the radix cudramiae (spina pulchroides) decoction pieces are decocted according to the scheme to prepare the standard radix cudramiae (spina pulchroides) decoction, the average transfer rate of the taxifolin is 23.73 percent, the measured transfer rate range is 13.32 to 38.68 percent, and the SD is 8.01. According to technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, the allowable range of the content transfer rate of the taxifolin is 16.61-30.85 percent calculated according to 70-130 percent of the mean value of the transfer rate; the content of the compound is 7.71-47.76% in terms of-2 SD and +3 SD. The results show that the taxifolin transfer rate in 15 batches of standard decoction is within the allowable range of-2 SD and +3 SD.
The average content of the taxifolin in the product is 29.08mg/g, the measured content range is 4.97-54.18 mg/g, and the SD is 18.01; calculated according to the mean value +/-3 SD, the allowable range of the content of the taxifolin is-24.95-83.11 mg/g. Since-3 SD is a negative value, the lowest value of the measured values, 4.97, is the lower limit. Therefore, the total content range of the taxifolin in the standard decoction is drawn as follows: 5.0 to 83.0 mg/g. The results show that the total content of the taxifolin in 15 batches of standard decoction and the transfer rate thereof are within the allowable range, and can provide reference basis for the quality research of the formula granules of the cudrania cochinchinensis (spina gleditsiae).
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A quality control method of a radix cudraniae standard decoction is characterized in that the quality control method is to draw up the content range of the taxifolin of the standard decoction to be 5.0-83.0mg/g through the characteristics, the dry extract extraction rate, the thin layer identification, the extract, the characteristic map and the taxifolin content measurement of the radix cudraniae standard decoction; wherein, the extraction rate of the dry extract is measured by adopting a decoction method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the taxifolin content are determined by liquid chromatography.
2. The quality control method of the radix cudraniae standard decoction according to claim 1, wherein the limit range of the dry extract yield is 4.0-7.5%.
3. The quality control method of the radix cudramiae standard decoction according to claim 1, which is characterized in that the thin-layer chromatography comprises the following steps:
a1, adding methanol into the radix Cudraniae decoction, performing ultrasonic treatment, filtering, evaporating the filtrate, and dissolving the residue in methanol to obtain a sample solution a;
a2, decocting radix Cudraniae control material, filtering, evaporating filtrate, and dissolving residue in methanol to obtain control solution a;
a3, setting thin-layer chromatography conditions: silica gel G thin layer plate; sample amount of spotting: the solution a of the test sample is 5ul, and the solution a of the reference medicinal material is 10 ul; developing agent: mixing dichloromethane, ethyl acetate and methanol in a volume ratio of 12:5: 2; and (5) placing under an ultraviolet lamp for inspection.
4. The method of claim 1, wherein the hot dipping method comprises ethanol as solvent, and the extract content is not less than 46.5% as measured by hot dipping method in alcohol-soluble extract measurement method.
5. The quality control method of radix Cudraniae standard decoction according to claim 1, wherein the characteristic spectrum and the method for measuring the content of taxifolin comprise respectively extracting radix Cudraniae reference solution b and test solution b, injecting into a liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are as follows: a chromatographic column: c18; mobile phase: performing gradient elution by using methanol as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; flow rate: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 290 nm.
6. The quality control method of the Chuanbro stone standard decoction according to claim 5, characterized in that the test solution b is prepared by collecting a Chuanbro stone standard decoction sample, adding 25 times of 70% methanol, sealing, subjecting to ultrasound, cooling, weighing, supplementing with 70% methanol, reducing weight loss, shaking, and filtering.
7. The method for controlling the quality of the cudrania cochinchinensis standard decoction according to claim 5, wherein the method for measuring the characteristic spectrum by using the liquid chromatography further comprises the steps of injecting the test substances of the same batch into the liquid chromatograph, measuring, and evaluating the similarity of the established common characteristic peaks.
8. The quality control method of radix Cudraniae standard decoction according to claim 5, wherein the characteristic spectrum and the method for determining the content of taxifolin by liquid chromatography further comprises feeding samples of the same batch of samples for 0h, 3h, 6h, 9h, 12h, and 24h respectively, determining peak shape and peak number, and performing similarity evaluation on common characteristic peaks for testing the stability of the sample solution.
9. The quality control method of a radix Cudraniae standard decoction according to claim 5, wherein the determination of the characteristic spectrum and the content of taxifolin by liquid chromatography further comprises continuously feeding 6 samples of the same batch of samples to determine peak shape and peak number, and performing similarity evaluation on common characteristic peaks for testing whether the precision of a chromatograph is good.
10. The method for quality control of a standard decoction of radix Cudraniae according to claim 5, wherein the method for determining the content of taxifolin by liquid chromatography further comprises the step of weighing a sample to be tested and adding the sample to a taxifolin reference solution to test the average sample recovery rate of taxifolin.
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