CN117434200A - Quality detection method for spora Lygodii formula particles - Google Patents
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- 239000002245 particle Substances 0.000 title claims abstract description 74
- 238000001514 detection method Methods 0.000 title claims abstract description 28
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Classifications
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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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- 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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a quality detection method of spora Lygodii formula particles, which comprises the steps of carrying out thin-layer identification, characteristic spectrum construction and caffeic acid content measurement on the spora Lygodii formula particles, limiting the content standard of the formula particles to 3.0-11.3 mg of caffeic acid in each 1g, wherein the thin-layer identification is carried out by adopting a thin-layer chromatography, and the characteristic spectrum construction and the caffeic acid content measurement are both carried out by adopting a liquid chromatography. According to the quality detection method for the spora lygodii formula particles, the quality of the spora lygodii formula particles is assessed through thin-layer identification, feature map construction and detection of caffeic acid content measurement, so that the inherent quality of the formula particles can be comprehensively reflected, and the quality of the spora lygodii formula particles can be better controlled.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality detection method of spora Lygodii formula particles.
Background
Modern medicines are required to have three characteristics of stability, uniformity, safety and effectiveness, and for Chinese patent medicines, various means are required to be adopted for detection, so that the reliability and stability of detection results are ensured. The traditional Chinese medicine formula granule is a single traditional Chinese medicine product prepared by adopting modern scientific technology and imitating the traditional Chinese medicine decoction decocting way, and refining the traditional Chinese medicine decoction pieces through the processes of leaching, concentrating, drying and the like. The product maintains the property, taste and efficacy of the traditional Chinese medicine decoction pieces, has stable quality, is applied to the formulation of clinical prescriptions of the traditional Chinese medicine, meets the requirements of dialectical treatment and prescription change, and has the advantages of no need of decoction, convenient administration, quick absorption, accurate dosage, safety, cleanness, convenient carrying and the like. The single medicinal materials are subjected to water extraction, concentration, drying, granulation and other procedures to prepare the formula particles, and compared with the traditional medicinal materials, the form characteristics of the formula particles are obviously changed, and the authenticity and quality of the product cannot be obtained by naked eye observation. Therefore, it is necessary to establish a quality evaluation system of the traditional Chinese medicine formula particles so as to comprehensively reflect the inherent quality of the formula particles. The spora Lygodii is dried mature spore of Lygodium japonicum (thunder.) Sw. of Lygodiaceae. Harvesting vine leaves when the spores are not shed in autumn, sun-drying, kneading or beating spores, and removing the vine leaves. Sweet and salty in taste, cold in nature; enter the bladder and small intestine channels; clearing away damp-heat, treating stranguria and relieving pain; is mainly used for treating heat stranguria, stranguria with stone, stranguria with blood, stranguria with paste, and painful urethra. At present, a systematic quality detection method is not formed on the spora lygodii formula particles, and the existing detection means are only adopted to detect the spora lygodii formula particles, so that the whole internal quality of the spora lygodii formula particles cannot be reflected, and the quality control requirement of the traditional Chinese medicine formula particles cannot be met. Therefore, it is necessary to establish a quality detection method of spora Lygodii formula particles for controlling the quality of medicinal materials.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a spora lygodii formula particle quality detection method which comprehensively reflects the internal quality of formula particles and better controls the quality of the formula particles.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
a quality detection method of spora Lygodii formula particles comprises the following steps:
carrying out thin-layer identification, characteristic spectrum construction and caffeic acid content measurement on the spora lygodii formula particles, limiting the content standard of the formula particles to 3.0-11.3 mg of caffeic acid in each 1g of the formula particles, wherein the thin-layer identification is carried out by adopting a thin-layer chromatography, and the characteristic spectrum construction and the caffeic acid content measurement are both carried out by adopting a liquid chromatography;
the determination of the characteristic spectrum by liquid chromatography comprises: taking a solution prepared from a climbing fern spore reference medicinal material as a reference substance solution a of the reference medicinal material, taking a solution prepared from a caffeic acid reference substance as a reference substance solution a, taking a solution prepared from climbing fern spore formula particle samples as a test substance solution a, respectively precisely sucking the reference substance solution a of the reference medicinal material, the reference substance solution a of the reference substance and the test substance solution a, respectively injecting the reference substance solution a, the reference substance solution a and the test substance solution a into a liquid chromatograph, and measuring to obtain the climbing fern spore extract; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; mobile phase: using methanol as a mobile phase A and 0.05% 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
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0～25 | 13→30 | 87→70 |
| 25～34 | 30→34 | 70→66 |
| 34～40 | 34 | 66 |
| 40～45 | 34→13 | 66→87 |
Flow rate: 1.0mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 225nm.
In one embodiment, the thin layer chromatography comprises the steps of:
(1) Preparing a test sample solution b: taking 0.3g of spora Lygodii formula granule powder, adding 25ml of methanol, performing ultrasonic treatment for 30 min, cooling, filtering, evaporating filtrate to dryness, and adding 0.5ml of methanol into residue to dissolve to obtain a sample solution b;
(2) Preparing a control medicinal material solution b: taking 0.3g of climbing fern spore as reference medicine, adding 50ml of water, decocting and keeping a micro-boiling state for 30 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of methanol into the residue, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating the filtrate to dryness, and adding 0.5ml of methanol into the residue to dissolve to obtain a reference medicine solution b;
(3) Thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: polyamide thin layer boards; sample application amount: the sample solution b and the control medicinal material solution b are 5 μl respectively; developing agent: taking methanol-glacial acetic acid-water with the volume ratio of 4:1:5 as a developing agent, developing, taking out, airing, spraying an aluminum trichloride test solution, airing, and inspecting under a 365nm ultraviolet lamp.
In one embodiment, the determination of the characteristic spectrum by liquid chromatography further comprises the steps of:
(1) Preparing a reference substance solution a of a control medicinal material: taking 0.8g of climbing fern spore reference medicine, adding 20ml of 10% methanol, heating and refluxing for 40 minutes, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as reference medicine reference solution a;
(2) Preparing a reference substance solution a of a reference substance: taking a proper amount of caffeic acid reference substance, precisely weighing, adding methanol to prepare a solution containing 50 mug per 1ml, and taking the solution as reference substance solution a;
(3) Preparing a test sample solution a: taking proper amount of spora Lygodii formula particles, grinding, taking 0.2g, placing into a conical flask with a plug, adding 80% methanol 20ml, performing ultrasonic treatment with power of 250W and frequency of 40kHz for 20 minutes, cooling, shaking, filtering, and taking the subsequent filtrate as a sample solution a.
In one embodiment, the chromatogram of the sample measured by liquid chromatography shows 6 characteristic peaks, and the retention time of the 6 characteristic peaks corresponds to that of the chromatogram of the reference material; wherein, the retention time of the peak 3 corresponds to that of the reference substance of the caffeic acid, the peak corresponding to the reference substance of the caffeic acid is taken as an S peak, and the relative retention time of the peak 1, the peak 2, the peak 4, the peak 5, the peak 6 and the S peak in the chromatogram of the sample to be tested is calculated, and the relative retention time is within +/-10% of a specified value; the specified value is: peak 1 relative retention time 0.63, peak 2 relative retention time 0.95, peak 4 relative retention time 1.22, peak 5 relative retention time 1.43, peak 6 relative retention time 1.56.
In one embodiment, measuring caffeic acid content using liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from caffeic acid reference substance as a reference substance solution c, taking a solution prepared from spora Lygodii formula particle samples 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 product; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; mobile phase: the volume ratio is 21:79 methanol-0.05% phosphoric acid solution as mobile phase; detection wavelength: 320nm.
In one embodiment, the determination of caffeic acid content by liquid chromatography further comprises the steps of:
(1) Preparing a reference substance solution c: taking a proper amount of caffeic acid reference substance, precisely weighing, adding methanol to prepare a solution containing 50 mug of caffeic acid per 1ml, and taking the solution as reference substance solution c;
(2) Preparing a test sample solution c: taking proper amount of spora Lygodii formula particles, grinding, taking 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 20ml of 80% methanol, sealing, weighing, performing ultrasonic treatment with power of 250W and frequency of 40kHz for 20 minutes, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.
In one embodiment, the method further comprises: performing character identification, dry extract extraction rate inspection and extract inspection on the spora Lygodii formula particles, wherein the extract inspection is measured by adopting a hot dipping method.
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.
Compared with the prior art, the quality detection method for the spora Lygodii formula particles has the beneficial effects that: the quality of the spora Lygodii formula particles is evaluated by carrying out thin-layer identification, feature map construction, caffeic acid content measurement and other multi-aspect measurement on the spora Lygodii formula particles, a solid foundation is laid for the stability of the quality of products, a feasible quality standard of the spora Lygodii formula particles can be established, the quality of the spora Lygodii formula particles is effectively controlled, and the chromatographic conditions are adopted for liquid phase analysis, so that the feature map with better and clearer separation degree can be obtained, and the method has the advantages of better stability, high precision and better reproducibility. The climbing fern spore decoction pieces are decocted to prepare climbing fern spore decoction piece formula granules, and the caffeic acid content range is 3.0-11.3 mg/g is formulated according to the technical requirements of quality control and standard formulation of traditional Chinese medicine formula granules.
Drawings
FIG. 1 is a thin-layer identification map of spora Lygodii decoction piece standard decoction pieces; wherein A: negative sample, S: 1 to 15 percent of climbing fern spore as reference medicinal material: sample 15 batches of standard soup samples.
FIG. 2 is a graph showing a fit of 18 batches of spora Lygodii standard decoction to a control drug; wherein, peak 1: protocatechuic aldehyde, peak 3 (S): caffeic acid, peak 5: 4-coumaric acid; chromatographic column: SPOLAR C18S 5,4.6 mm. Times.250 mm,5 μm.
FIG. 3 is a TLC chart of thin-layer identification of batch production samples of spora Lygodii decoction piece formula particles 3 according to an embodiment of the present invention; wherein, the group A is a negative sample solution, the group S is a control medicinal material solution, and the group 1-3 are test samples ZS 221101-ZS 221103.
Fig. 4 shows HPLC feature patterns of different extraction methods in investigation of feature pattern measurement extraction method according to an embodiment of the present invention.
Fig. 5 shows HPLC profiles at different extraction times in a profile measurement extraction time investigation according to an embodiment of the present invention.
Fig. 6 is a graph showing HPLC profiles of different extraction solvents in a profile measurement of extraction solvents under investigation in accordance with an embodiment of the present invention.
FIG. 7 is a graph showing HPLC profiles of different sample volumes in a profile measurement sample volume investigation according to an embodiment of the present invention.
FIG. 8 is a graph showing HPLC profiles of different solutions in a specificity study for profile determination in accordance with one embodiment of the present invention.
FIG. 9 is a graph of the superposition of peaks common to stability tests in a characteristic spectrum measurement according to an embodiment of the present invention.
Fig. 10 is a superposition of 3 batches of finished product feature maps according to an embodiment of the invention.
Fig. 11 is a superposition diagram of characteristic maps of the spora Lygodii 3 batches of finished products and the reference substance of the reference medicinal material in an embodiment of the invention.
FIG. 12 is a graph showing a peak-fitting pattern shared by the spora Lygodii product and the characteristic patterns of the reference medicinal materials in an embodiment of the present invention; wherein peak 1 is protocatechuic acid; peak 3 (S) is caffeic acid; peak 5 is 4-coumaric acid; chromatographic column: SPOLAR C18S 5,4.6 mm. Times.250 mm,5 μm.
FIG. 13 is a graph showing comparison of blank solvents in a content determination specificity study according to an embodiment of the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Regarding the quality standard of the spora Lygodii standard decoction:
the product is a standard decoction prepared by processing dried mature spores of Lygodium japonicum (thunder.) Sw. of Lygodiaceae and referring to the medical institution Chinese medicine decoction room management Specification.
[ PREPARATION METHOD ] taking spora Lygodii decoction pieces, referring to "medical institution Chinese medicine decoction Chamber management Specification", fixing pretreatment method, decocting times, water amount, and decoction time, decocting, performing solid-liquid separation, concentrating, drying, and pulverizing.
[ paste yield ] 18 batches of spora Lygodii decoction pieces are taken, 18 batches of standard decoction dry paste powder are prepared according to the preparation method, the dry extract (paste yield) yield is calculated by using the dry paste powder (see table I), the average yield is calculated to be 7.028%, the paste yield allowable range is calculated according to the standard decoction paste yield allowable range (average value 70% -130%), the paste yield allowable range is 4.92% -9.14%, and the sea Jin Shagan extract paste yield allowable range is 5.0% -9.0%.
Table I paste yield of climbing fern spore decoction piece standard decoction
The results show that the paste yield of 18 batches of standard decoction is 5.8-8.7%, and the paste yield of 18 batches accords with the range of 5.0-9.0% of the planned limit.
[ PROTECTS ] according to the physical character characteristics of 18 batches of standard decoction, the decoction is described as brown-yellow to brown-brown powder; light smell and bland taste.
The thin-layer identification method is established by referring to the method under the "spora Lygodii" item of Chinese pharmacopoeia 2020 edition and taking the spora Lygodii reference medicine as a reference. Through 15 batches of standard decoction samples, the spots of the test sample are clear, and the negative control sample has no interference, so the method is listed in the standard decoction quality standard text [ identification ] of the spora Lygodii formula granule.
Taking 0.3g of the product powder, adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution. 1ml of methanol solution was taken as a negative sample solution. Taking 0.3g of climbing fern spore as reference medicine, adding 50ml of water, decocting and keeping a micro-boiling state for 30 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of methanol into the residue, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating the filtrate to dryness, and adding 0.5ml of methanol into the residue to dissolve the residue to obtain a reference medicine solution. According to thin layer chromatography (rule 0502 of four parts of the 2020 edition of Chinese pharmacopoeia), 5 μl of each of the sample solution and the control medicinal solution is absorbed, and the sample solution and the control medicinal solution are respectively spotted on the same polyamide thin layer plate, and are spread with methanol-glacial acetic acid-water (4:1:5) as developing agent, taken out, dried, sprayed with aluminum trichloride test solution, dried, and then detected under an ultraviolet lamp (365 nm). As shown in FIG. 1, fluorescent spots of the same color appear on the chromatogram of the test sample at positions corresponding to those of the chromatogram of the control drug.
[ characteristic map ] is measured by high performance liquid chromatography (general rule 0512 in the year 2020).
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (SPOLAR C18S 5, column length of 250mm, inner diameter of 4.6mm, particle size of 5 μm); using methanol as a mobile phase A and 0.05% phosphoric acid solution as a mobile phase B, and performing gradient elution according to the specification in a table II; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 225nm. The theoretical plate number should be not less than 5000 as calculated by caffeic acid peak.
Table II gradient elution procedure
Preparation of reference solution: taking 0.8g of spora Lygodii as reference material, adding 10% methanol 20ml, heating and refluxing for 40 min, cooling, shaking, filtering, and collecting the subsequent filtrate as reference material solution. And taking a proper amount of caffeic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing 80 mug per 1ml as a reference substance solution of the reference substance.
Preparation of test solution: about 0.2g of fine powder (batch number: Y221007 PT) of the Japanese climbing fern Sha Biaozhun decoction is taken, placed in a conical flask with a plug, added with 20ml of 80% methanol, subjected to ultrasonic treatment (power 300W, frequency 40 kHz) for 20 minutes, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain the Chinese medicinal decoction.
Assay: precisely sucking reference solution of reference material, reference solution of reference material and sample solution of test material respectively by 10 μl, and measuring with liquid chromatograph.
According to the above-mentioned established characteristic spectrum analysis method, the characteristic spectrum of 18 batches of spora Lygodii standard decoction is measured, and the result analysis is shown in figure 2, and 6 common characteristic peaks are calibrated, wherein the peak 3 is caffeic acid. Calculating the relative retention time of the other 5 characteristic peaks by taking the peak corresponding to the caffeic acid reference substance as an S peak, and respectively setting the average value of the relative retention time of 18 batches of sample peaks as a specified value to be: 0.60 (Peak 1), 0.94 (Peak 2), 1.28 (Peak 4), 1.46 (Peak 5), 1.62 (Peak 6), and the relative retention time allowable range was assumed to be.+ -. 10% taking into consideration multi-factor errors of test operation, instrument, reagent, etc.
Taking 18 batches of standard decoction, taking ethanol as a solvent according to the technical requirement, and measuring according to a hot dipping method under the condition of alcohol-soluble extract measurement method (general rule 2201 in the year 2020 edition of Chinese pharmacopoeia). The result shows that the average value of the 18 batches of standard decoction extracts is 36.514 percent, and the alcohol-soluble extract of the product is not less than 25.6 percent by referring to the lower limit of the allowable range of the standard limit (average value 70-130 percent). The measurement results of the 18 batches of standard decoction meet the requirements of the planned limit.
[ MEANS FOR SOLVING ] according to high performance liquid chromatography (rule 0512 in the year 2020 edition of Chinese pharmacopoeia).
Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica (Shimadzu Shim-pack GIST C18,4.6 mm. Times.250 mm,5 μm) was used as a filler; methanol-0.05% phosphoric acid solution (21:79) is taken as a mobile phase; the detection wavelength was 320nm. The theoretical plate number should be not less than 5000 as calculated by caffeic acid peak.
Preparation of a control solution: taking a proper amount of caffeic acid reference substance, precisely weighing, adding methanol to prepare a solution containing 50 mug per 1 ml.
Preparation of test solution: about 0.2g of fine powder (batch number: Y221007 PT) of the Japanese sea gold Sha Biaozhun decoction is precisely weighed, placed in a conical flask with a plug, precisely added with 20ml of 80% methanol, sealed, weighed, subjected to ultrasonic treatment (power 300W, frequency 40 kHz) for 20 minutes, cooled, weighed again, complemented with 80% methanol to reduce the weight, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the Chinese medicinal decoction.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
The allowable range of caffeic acid content of the standard decoction is as follows: 5.9 mg/g-12.6 mg/g.
[ storage ] seal.
Further, the embodiment provides a quality detection method of spora Lygodii formula particles, which is characterized in that the characteristics, dry extract yield, thin layer identification, characteristic spectrum, extract and caffeic acid content of the spora Lygodii formula particles are measured, the content standard of the spora Lygodii formula particles is limited to 3.0-11.3 mg of caffeic acid in each 1g, wherein the thin layer identification is carried out by adopting a thin layer chromatography, the characteristic spectrum and the caffeic acid content are measured by adopting a liquid chromatography, and the extract is measured by adopting a hot dipping method.
Preparing spora Lygodii formula particles: the product is prepared from a part of Japanese climbing fern spore traditional Chinese medicinal material under the item and medicinal parts according to the technical requirement in the 'Chinese pharmacopoeia' 2020 edition, and comprises the following medicinal material plant parts of the family name, chinese name, latin and medicinal parts: the product is prepared from dried mature spore of Lygodium japonicum (thunder.) Sw. belonging to family Lygodiaceae by processing and processing according to main quality index of standard decoction.
The specific preparation method comprises the following steps: 10000g of climbing fern spore decoction pieces are taken, water is added for decoction, filtration is carried out, the filtrate is concentrated into clear paste (the paste yield of the dry extract is 5.0% -9.0%), a proper amount of auxiliary materials are added for drying (or drying and crushing), a proper amount of auxiliary materials are added, and the mixture is evenly mixed, granulated and made into 1000g, thus obtaining the climbing fern spore decoction pieces.
According to the paste yield of 18 batches of standard decoction, the range of 70-130% of the average value of the paste yield is defined as the dry extract paste yield, and the dry extract paste yield is 5.0-9.0%.
Inspection of spora Lygodii formula granule methodology:
1. property investigation
Traits: taking one batch of three pilot samples of the spora Lygodii formula particles, observing and recording the form, color and smell of the samples, and simultaneously combining the color and smell of the standard soup samples. The product is granule, and is brown to brown; light smell and bland taste.
2. Thin layer authentication
The thin-layer identification method is established by taking the spora lygodii reference medicinal material as a reference, and the chromatographic spots of the test sample are clear and the negative reference sample is undisturbed, so the method is listed in the text [ identification ] of the quality standard of the spora lygodii formula particles.
Specifically, the thin layer identification method of the spora Lygodii formula particle comprises the following steps:
sample solution preparation: taking 0.3g of the product powder, adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
Negative sample solution preparation: a negative sample solution was prepared by the same method as that for 0.3g of maltodextrin.
Preparing a control medicinal material solution: taking 0.3g of climbing fern spore as reference medicine, adding 50ml of water, decocting and keeping a micro-boiling state for 30 minutes, filtering, evaporating filtrate to dryness, adding 25ml of methanol into residues, and preparing the reference medicine solution by the same method.
According to thin layer chromatography (rule 0502 of four parts of the 2020 edition of Chinese pharmacopoeia), 5 μl of each of the sample solution and the control medicinal solution is absorbed, respectively spotted on the same polyamide thin layer plate, and developed with methanol-glacial acetic acid-water (4:1:5) as developing agent, taken out, air-dried, sprayed with aluminum trichloride test solution, air-dried, and inspected under ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material.
According to the detection method, 3 batches of production sample test sample solution, negative sample (maltodextrin) solution and control medicinal material solution are taken, spotted, and thin-layer identification is carried out. As shown in FIG. 3, the results show that spots with the same color appear on the sample chromatogram at the positions corresponding to the control chromatogram and the control chromatogram.
3. Feature map
3.1 reagents and reagents
3.1.1 reagent ethanol (Tianjin far chemical reagent Co., ltd.), methanol (Tianjin Density European chemical reagent Co., ltd.), acetonitrile (Tianjin Density European chemical reagent Co., ltd.) chromatography; the water is ultrapure water (self-made in laboratory); phosphoric acid (Tianjin, denou chemical Co., ltd.); glacial acetic acid (Tianjin far chemical agent Co., ltd.); formic acid (Tianjin, denou chemical Co., ltd.).
3.1.2 control and control caffeic acid (lot number: 110885-201603, content: 99.7%), spora Lygodii control (lot number: 121607-201202, national institute of food and drug testing).
3.2 feature map measuring method
3.2.1 chromatographic conditions and System applicability experiments
Octadecylsilane chemically bonded silica (SPOLAR C18S 5 (4.6 mm. Times.250 mm,5 μm)) as filler; gradient elution was performed as specified in table 1 with methanol as mobile phase a and 0.05% phosphoric acid solution as mobile phase B; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 225nm. The theoretical plate number should be not less than 5000 as calculated by caffeic acid peak.
TABLE 1 gradient elution procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0～25 | 13→30 | 87→70 |
| 25～34 | 30→34 | 70→66 |
| 34～40 | 34 | 66 |
| 40～45 | 34→13 | 66→87 |
3.2.2 preparation of reference solution
Taking 0.8g of spora Lygodii as reference material, adding 10% methanol 20ml, heating and refluxing for 40 min, cooling, shaking, filtering, and collecting the subsequent filtrate as reference material solution.
And (3) taking a proper amount of caffeic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing 50 mug of each 1ml of the reference substance as a reference substance solution of the reference substance.
3.2.3 preparation of sample solutions
Grinding the materials, taking about 0.2g, placing into a conical flask with a plug, adding 80% methanol 20ml, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 20 min, cooling, shaking, filtering, and collecting the filtrate.
3.2.4 assays
Precisely sucking reference solution of reference material, reference solution of reference material and sample solution of test material respectively by 10 μl, and measuring with liquid chromatograph.
3.3 methodology investigation of feature atlas determination
3.3.1 investigation of extraction method
Test solutions were prepared by different extraction methods, and the measurement was performed according to the method described under "3.2" above. As shown in FIG. 4 and Table 2, the results show that the main chromatographic peaks of the sample solutions with different extraction modes are consistent, the peak shape difference is not obvious, and the RSD values are less than 3%, but the ultrasonic extraction effect is obviously better than that of the reflux method, so that the ultrasonic extraction is selected as the sample extraction mode.
TABLE 2 comparison of extraction methods
3.3.2 extraction time investigation
Test solutions were prepared at different times, and the measurement was performed according to the method described under "3.2" above. As shown in FIG. 5 and Table 3, the results show that the main chromatographic peaks of the sample solutions at different extraction times are consistent, the peak shape difference is not obvious, and the RSD values are less than 3%, but the extraction effect of 20 minutes of extraction is complete, so that the extraction time of the sample is determined to be 20 minutes.
TABLE 3 comparison of extraction times
3.3.3 investigation of extraction solvent
Test solutions were prepared with different extraction solvents, respectively, and the measurement was performed according to the method described under item "3.2" above. As shown in fig. 6 and table 4, the results showed that the extraction effect was good when 80% methanol was extracted as the solvent, and finally, 80% methanol was determined as the solvent.
TABLE 4 comparison of extraction solvents
3.3.4 investigation of sample size
Test solutions were prepared in different amounts and were measured as described under "3.2" above. As shown in FIG. 7 and Table 5, the results revealed that the number of main peaks was uniform and that the extraction effect was optimal when the sample weighing amount of the extracted sample was 0.2g, so that the sample weighing amount of the extracted sample was 0.2g.
TABLE 5 comparison of sample volumes
In summary, the main parameters of the method for preparing the sample solution are determined as follows: grinding the above materials, collecting about 0.2g, placing into conical flask with plug, adding 80% methanol 20ml, ultrasound (power 250W, frequency 40 kHz) for 20 min, cooling, shaking, filtering, and collecting filtrate.
3.4 feature map analysis method verification
3.4.1 specificity investigation
Taking a proper amount of maltodextrin for a test sample, injecting 10 μl according to the test sample treatment mode, and measuring under the chromatographic condition of 3.2. The results show that: the blank solvent is free of interference, and the method has good specificity, as shown in figure 8.
3.4.2 stability test
A batch of about 0.2g of the sample was sampled and assayed by the test method under item "3.2" at 0h, 2h, 4h, 8h, 12h, 24h, respectively, and the peak shape and number were substantially stable (see FIG. 9). And the similarity evaluation system (2012 edition) of the traditional Chinese medicine chromatographic fingerprint chart is adopted to evaluate the similarity of the designated 6 common characteristic peaks, the relative retention time and the RSD of the relative peak area are all in the qualified range, the detail is shown in tables 6-7, and the test shows that the sample solution is stable within 24 hours.
TABLE 6 stability test characterization profile versus retention time
| Peak number | 0h | 2h | 4h | 8h | 12h | 24h | RSD(％) |
| 1 | 0.625 | 0.625 | 0.624 | 0.624 | 0.621 | 0.624 | 0.24 |
| 2 | 0.946 | 0.945 | 0.945 | 0.944 | 0.940 | 0.944 | 0.22 |
| 3(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 1.219 | 1.218 | 1.217 | 1.215 | 1.238 | 1.216 | 0.71 |
| 5 | 1.429 | 1.429 | 1.429 | 1.427 | 1.426 | 1.428 | 0.09 |
| 6 | 1.563 | 1.562 | 1.562 | 1.559 | 1.558 | 1.561 | 0.12 |
TABLE 7 stability test characteristic spectrum versus peak area
3.4.3 knots
The characteristic spectrum method meets the requirements through the investigation of specificity and stability, and the results show that the established method can be well used for measuring the characteristic spectrum of the spora Lygodii formula particles.
3.5 characterization analysis of three batches of finished product feature patterns
3.5.1 3 batches of finished product characteristic spectrum measurement
According to the proposed characteristic spectrum analysis method, 3 batches of mass production sample characteristic chromatograms are measured, and the result shows that 6 common peaks exist in the three batches of mass production sample characteristic chromatograms and correspond to the retention time of 6 characteristic peaks in the reference object chromatogram of the reference medicinal material, wherein the retention time of the peak 3 corresponds to the retention time of the reference object peak of the caffeic acid reference object, and the common peak characteristic chromatograms are shown in fig. 10-12 in detail.
3.5.2 3 evaluation of characteristic chromatograms of formulation particles
And (3) performing similarity evaluation on the characteristic patterns of 3 batches of mass production samples by adopting a traditional Chinese medicine chromatographic fingerprint pattern similarity evaluation system (2012 edition), and calibrating 6 common characteristic peaks, wherein the peak 3 is a characteristic peak of a caffeic acid reference substance. The relative retention times of characteristic peaks 1, 2, 4, 5 and 6 were calculated using the corresponding peak of caffeic acid control as the S peak, and are shown in table 8.
TABLE 8-3 peak to hold time for batch product sharing
| Peak number | Finished product 1 | Finished product 2 | Finished product 3 | Mean value of | Relative reservationTime range + -10% |
| 1 | 0.627 | 0.627 | 0.627 | 0.627 | 0.564～0.690 |
| 2 | 0.948 | 0.947 | 0.947 | 0.947 | 0.853～1.042 |
| 3(S) | 1.000 | 1.000 | 1.000 | 1.000 | / |
| 4 | 1.224 | 1.223 | 1.223 | 1.223 | 1.101～1.346 |
| 5 | 1.43 | 1.431 | 1.432 | 1.431 | 1.288～1.574 |
| 6 | 1.564 | 1.564 | 1.566 | 1.565 | 1.408～1.721 |
In conclusion, the characteristic spectrum detection method of the product formula granule is established by adopting a high performance liquid chromatography. The characteristic spectrum of 3 batches of mass production samples is evaluated, 6 common characteristic peaks are calibrated, wherein the peak 3 corresponds to the retention time of the reference peak of the caffeic acid reference substance, the peak corresponding to the caffeic acid reference substance is taken as an S peak, the relative retention time of the peak 1, the peak 2, the peak 4, the peak 5, the peak 6 and the S peak is calculated, and the average value of the relative retention time of the characteristic peaks of the 3 batches of mass production samples is set as a specified value, and the specified values are respectively: 0.63 (Peak 1), 0.95 (Peak 2), 1.22 (Peak 4), 1.43 (Peak 5), 1.56 (Peak 6), combined with standard decoction, pilot and scale production test results and test errors to determine the relative retention time of each peak allowed range of + -10%.
4 test item test
4.1 checking the general rule of granules according to the related requirements under the general rule of 0104 granules of the fourth edition of the Chinese pharmacopoeia 2020, checking the granularity, the moisture and the dissolubility of the middle samples (batch numbers: ZS221101, ZS221102 and ZS 221103), and checking the microorganism limit and the difference of the loading quantity, wherein the related requirements under the general rule of 0104 granules of the fourth edition of the Chinese pharmacopoeia 2020 are met, and the table 9 is shown.
Table 9-inspection of scale production samples of spora Lygodii formulation particles
| Inspection item | ZS221101 | ZS221102 | ZS221103 |
| Moisture (less than or equal to 8.0 percent) | 5.0％ | 5.2％ | 5.1％ |
| Particle size (less than or equal to 15 percent) | 5％ | 6％ | 5％ |
| Solubility of | Meets the regulations | Meets the regulations | Meets the regulations |
| Difference in loading | Meets the regulations | Meets the regulations | Meets the regulations |
| Microbial limitation | Meets the regulations | Meets the regulations | Meets the regulations |
According to the rule 0104 granule item of the general rule 2020 of Chinese pharmacopoeia, each batch number is obtained by taking 1 bag of the test sample, respectively heating 200ml of water, stirring for 5 minutes, immediately observing, and completely dissolving the test sample, wherein no coke dust and slight turbidity are generated, and all the test sample meets the related requirements of solubility.
4.2 exogenous toxic and harmful substance detection
The medicinal materials and decoction pieces are collected in the first part of the 2020 edition of Chinese pharmacopoeia, and related harmful substances are not controlled in the standard. Exogenous toxic and harmful substances are not listed as examination items.
5 extract
5.1 test methods
The extraction solvent of the traditional Chinese medicine formula granule is water for pharmacy, ethanol is used as solvent according to the technical requirement, and the solvent is measured according to a hot dipping method under the condition of alcohol-soluble extract measurement method (2201 in the general rule of the year 2020 of Chinese pharmacopoeia).
5.2 investigation of extract
About 2g each of 3 pilot samples was taken and ground and measured according to the law, the results are shown in Table 10.
TABLE 10 results of extract investigation
The mean extract of three pilot samples was 16.33%. According to the specification of the product (each 1g of formula particle corresponds to 10g of decoction pieces), the allowable range of extract=25.6% ×5.0% ×10×100% =12.8% is calculated by referring to the lower limit of the range of extract from 18 batches of standard decoction pieces (25.6%), and the lower limit of the range of extract yield from 5.0% to 9.0%, and if the allowable range of extract from the product is calculated as 70% of the standard decoction piece (25.6%), the allowable range of extract from the product is 25.6% ×0.7=17.92%, and the alcohol-soluble extract from the product is not less than 13.0% by comprehensive consideration. The extracts of the pilot samples are all more than 13.0 percent, which meets the requirements.
6 content determination
6.1 selection basis of the component to be measured
The spora Lygodii is dried mature spore of Lygodium japonicum (thunder.) Sw. of Lygodiaceae. The composition mainly has the effects of removing dampness and heat, treating stranguria and relieving pain and stopping bleeding, and the chemical components of the composition comprise brass, volatile oil, triterpene, glycoside, phenolic acid and the like, and researches show that the composition uses the benzoic acid represented by chlorogenic acid and caffeic acid as main components, so that caffeic acid is selected as a quality control quantitative standard of the spora lygodii formula particles.
6.2 method for measuring caffeic acid content
The measurement is carried out by high performance liquid chromatography (the rule 0512 of the edition of Chinese pharmacopoeia 2020).
Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica (Shimadzu Shim-pack GIST C18 (4.6 mm. Times.250 mm,5 μm)) is used as filler; methanol-0.05% phosphoric acid solution (21:79) is used as a mobile phase, and the detection wavelength is 320nm. The theoretical plate number should be not less than 5000 as calculated by caffeic acid peak.
Preparation of a control solution: taking a proper amount of caffeic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing 50 mug per 1 ml.
Preparation of test solution: grinding the product, taking about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 80% methanol 20ml, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 20 min, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking, filtering, and collecting the subsequent filtrate.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
6.3 inspection of spora Lygodii content measurement methodology
6.3.1 investigation of the extraction method
The sample preparation test solutions were extracted in different ways and assayed according to the method described under "6.2" above. The results show that ultrasonic extraction is superior to reflux extraction, so that the ultrasonic mode is selected to extract the sample, and the details are shown in Table 11.
TABLE 11 comparison of different extraction methods
6.3.2 investigation of extraction time
According to the method described in item 6.2, sample preparation test solutions were prepared by extracting samples at different times, respectively, and measurement was carried out according to law. The results showed that the extraction was substantially complete for 20 minutes, so the sample extraction time was determined to be 20 minutes, as detailed in Table 12.
TABLE 12 comparison of different extraction times
6.3.3 investigation of extraction solvent
According to the method described in item 6.2, test solutions were prepared by extracting samples with different solvents, respectively, and the measurement was carried out according to the law. The results showed that 80% methanol was the best solvent extraction, so 80% methanol was selected as the solvent extraction sample, as detailed in Table 13.
TABLE 13 comparison of different extraction solvents
6.3.4 sample size investigation
Test solutions were prepared in different amounts by the method described under "6.2" above, and the measurement was performed according to the law. The results showed that the extraction effect was better than the other two groups when the sampling amount was 0.2g, so that 0.2g was selected as the sampling amount for preparing the test sample solution, as shown in Table 14.
TABLE 14 comparison of different sample volumes
In summary, the main parameters of the method for preparing the sample solution are determined as follows: grinding the product into fine powder, taking about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 80% methanol 20ml, sealing, weighing, ultrasonically (power 250W, frequency 40 kHz) for 20 minutes, cooling, weighing again, supplementing the loss weight with 80% methanol, shaking, filtering, and collecting the subsequent filtrate.
6.4 content determination analysis method verification
6.4.1 specificity investigation
Maltodextrin was taken at about 0.1g and measured according to the chromatographic conditions set forth under item "6.2" above, and the test showed that: as shown in FIG. 13, maltodextrin blank was undisturbed and the process was well specific.
6.4.2 stability test
A batch of samples (batch number: ZS 221101) of about 0.2g was taken and sampled at 0h, 2h, 4h, 8h, 12h and 24h, respectively, according to the test method "6.2", the peak areas were measured, the RSD value of the caffeic acid content was calculated to be 0.16%, and the test showed that the test sample solution was stable within 24 hours, as shown in Table 15.
TABLE 15 stability test
6.5 knots
In conclusion, the whole analysis method is inspected by specificity and stability, and the result meets the requirements, so that the established method can be well used for measuring the content of the spora lygodii formula particles.
7 content limitation range establishment
7.1 measurement of the content of samples for three-batch Scale production the caffeic acid content of samples for 3-batch scale production was measured according to the above-described proposed content analysis method, and the results are shown in Table 16.
Table 16 results of three batch mass production sample determinations
According to the content range, the paste yield and the specification calculation of the climbing fern spore formula granule, the allowable range of caffeic acid content of the climbing fern spore formula granule is 5.9 mg/g-12.6 mg/g, and the allowable range of the paste yield of the formula granule is 5% -9%. The specification of the product is that each 1g of granule is equivalent to 10g of decoction pieces, and the dosage range of auxiliary materials is calculated according to the specification: maximum adjuvant dosage: 1-5% x 10 = 50%, minimum adjuvant usage: 1-9% x 10=10%, i.e. the dosage range of the auxiliary materials is 10% -50%. The content range of the formulation particles was thus calculated as: 5.9× (1-50%) =2.95 mg/g,12.6× (1-10%) =11.34 mg/g, i.e. the acceptable range of the content of the lygodium japonicum acid is 2.95mg/g to 11.34mg/g, and the content range of the product is 3.0mg/g to 11.3mg/g by combining the 3 batches of mass production samples.
The average content of three batches of mass production samples is 4.51mg/g, the actual measurement range is 4.45 mg/g-4.58 mg/g, and all the samples are in the planned range.
The 8 specifications are formulated according to the preparation method feeding amount and the process research preparation amount, namely, each 1g of formula particle is equivalent to 10g of decoction pieces.
9, the storage is formulated according to the basic requirements of variety storage.
Those skilled in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The quality detection method of the spora Lygodii formula particles is characterized by comprising the following steps of:
carrying out thin-layer identification, characteristic spectrum construction and caffeic acid content measurement on the spora lygodii formula particles, limiting the content standard of the formula particles to 3.0-11.3 mg of caffeic acid in each 1g of the formula particles, wherein the thin-layer identification is carried out by adopting a thin-layer chromatography, and the characteristic spectrum construction and the caffeic acid content measurement are both carried out by adopting a liquid chromatography;
the determination of the characteristic spectrum by liquid chromatography comprises: taking a solution prepared from a climbing fern spore reference medicinal material as a reference substance solution a of the reference medicinal material, taking a solution prepared from a caffeic acid reference substance as a reference substance solution a, taking a solution prepared from climbing fern spore formula particle samples as a test substance solution a, respectively precisely sucking the reference substance solution a of the reference medicinal material, the reference substance solution a of the reference substance and the test substance solution a, respectively injecting the reference substance solution a, the reference substance solution a and the test substance solution a into a liquid chromatograph, and measuring to obtain the climbing fern spore extract; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; mobile phase: using methanol as a mobile phase A and 0.05% 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: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 225nm.
2. The method for detecting the quality of spora Lygodii formula particles according to claim 1, wherein the thin-layer chromatography comprises the following steps:
(1) Preparing a test sample solution b: taking 0.3g of spora Lygodii formula granule powder, adding 25ml of methanol, performing ultrasonic treatment for 30 min, cooling, filtering, evaporating filtrate to dryness, and adding 0.5ml of methanol into residue to dissolve to obtain a sample solution b;
(2) Preparing a control medicinal material solution b: taking 0.3g of climbing fern spore as reference medicine, adding 50ml of water, decocting and keeping a micro-boiling state for 30 minutes, filtering, evaporating the filtrate to dryness, adding 25ml of methanol into the residue, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating the filtrate to dryness, and adding 0.5ml of methanol into the residue to dissolve to obtain a reference medicine solution b;
(3) Thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: polyamide thin layer boards; sample application amount: the sample solution b and the control medicinal material solution b are 5 μl respectively; developing agent: taking methanol-glacial acetic acid-water with the volume ratio of 4:1:5 as a developing agent, developing, taking out, airing, spraying an aluminum trichloride test solution, airing, and inspecting under a 365nm ultraviolet lamp.
3. The method for detecting the quality of spora Lygodii formula particles according to claim 1, wherein the characteristic spectrum is measured by adopting a liquid chromatography, further comprising the steps of:
(1) Preparing a reference substance solution a of a control medicinal material: taking 0.8g of climbing fern spore reference medicine, adding 20ml of 10% methanol, heating and refluxing for 40 minutes, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as reference medicine reference solution a;
(2) Preparing a reference substance solution a of a reference substance: taking a proper amount of caffeic acid reference substance, precisely weighing, adding methanol to prepare a solution containing 50 mug per 1ml, and taking the solution as reference substance solution a;
(3) Preparing a test sample solution a: taking proper amount of spora Lygodii formula particles, grinding, taking 0.2g, placing into a conical flask with a plug, adding 80% methanol 20ml, performing ultrasonic treatment with power of 250W and frequency of 40kHz for 20 minutes, cooling, shaking, filtering, and taking the subsequent filtrate as a sample solution a.
4. The method for detecting the quality of spora Lygodii formula particles according to claim 1, wherein 6 characteristic peaks are shown in a chromatogram of a test sample measured by liquid chromatography and correspond to 6 characteristic peak retention times in a chromatogram of a reference substance of a reference medicinal material; wherein, the retention time of the peak 3 corresponds to that of the reference substance of the caffeic acid, the peak corresponding to the reference substance of the caffeic acid is taken as an S peak, and the relative retention time of the peak 1, the peak 2, the peak 4, the peak 5, the peak 6 and the S peak in the chromatogram of the sample to be tested is calculated, and the relative retention time is within +/-10% of a specified value; the specified value is: peak 1 relative retention time 0.63, peak 2 relative retention time 0.95, peak 4 relative retention time 1.22, peak 5 relative retention time 1.43, peak 6 relative retention time 1.56.
5. The method for detecting the quality of spora Lygodii formula particles as claimed in claim 1, wherein the determination of the caffeic acid content by liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from caffeic acid reference substance as a reference substance solution c, taking a solution prepared from spora Lygodii formula particle samples 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 product; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; mobile phase: the volume ratio is 21:79 methanol-0.05% phosphoric acid solution as mobile phase; detection wavelength: 320nm.
6. The method for detecting the quality of spora Lygodii formula particles as claimed in claim 5, wherein the determination of the caffeic acid content by liquid chromatography further comprises the steps of:
(1) Preparing a reference substance solution c: taking a proper amount of caffeic acid reference substance, precisely weighing, adding methanol to prepare a solution containing 50 mug of caffeic acid per 1ml, and taking the solution as reference substance solution c;
(2) Preparing a test sample solution c: taking proper amount of spora Lygodii formula particles, grinding, taking 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 20ml of 80% methanol, sealing, weighing, performing ultrasonic treatment with power of 250W and frequency of 40kHz for 20 minutes, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.
7. The method for detecting the quality of spora Lygodii formula particles according to any one of the claims 1 to 6, further comprising a detection method,
performing character identification, dry extract extraction rate inspection and extract inspection on the spora Lygodii formula particles, wherein the extract inspection is measured by adopting a hot dipping method.
8. The method for detecting the quality of spora Lygodii formula particles as claimed in claim 7, wherein the hot dipping method uses ethanol as a solvent, and the range of the extract is measured by adopting a hot dipping method under the alcohol-soluble extract measuring method.
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