CN114689732B - Whole-process quality detection method in preparation of perilla leaf formula particles - Google Patents
Whole-process quality detection method in preparation of perilla leaf formula particles Download PDFInfo
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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
- G01N30/02—Column chromatography
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Medicines Containing Plant Substances (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a whole process quality detection method in preparation of perilla leaf formula particles, the perilla leaf formula particles and a preparation method thereof. The whole process quality detection method in the preparation of the perilla leaf formula granule comprises a quality detection method of perilla leaf decoction pieces, a quality detection method of perilla leaf intermediates and a quality detection method of the perilla leaf formula granule. The invention provides a new characteristic index and content index in quality detection, optimizes the index detection method, has strong specificity and good reproducibility, and can effectively control the quality of each link in the preparation process of the perilla leaf formula particles. The whole process quality detection method can ensure the high quality of the perilla leaf formula particles, has high product qualification rate, and can ensure small difference, good uniformity, high qualification rate, stable quality and high of the perilla leaf formula particles in the same production batch and among different production batches.
Description
Technical Field
The invention relates to traditional Chinese medicine decoction pieces, traditional Chinese medicine intermediates and traditional Chinese medicine formula granules, in particular to a whole process quality detection method for preparing perilla leaf formula granules, perilla leaf decoction pieces and a preparation method thereof, a perilla She Zhongjian body and a preparation method thereof, and perilla leaf formula granules and a preparation method thereof.
Background
The decoction is one of the earliest and most widely used dosage forms in China, but the decoction has the defects of complicated decoction process, inconvenient carrying, influence of decoction quality on the quality of traditional Chinese medicine decoction pieces and the decoction method of patients, poor quality control and the like, and solves the problems of quality stability, convenience and safety of the traditional Chinese medicine decoction. 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 high-quality traditional Chinese medicine formula particles can keep the taste and the efficacy of traditional Chinese medicine decoction pieces, are applied to the formulation of clinical prescriptions of traditional Chinese medicine, are suitable for the needs of dialectical treatment and prescription change, and have the advantages of no need of decoction, convenient administration, quick absorption, safety, cleanness, convenient carrying and the like. Although the traditional Chinese medicine formula particles have more advantages, people have doubts on the authenticity identification and the quality evaluation after the traditional Chinese medicine formula particles do not have the shape of decoction pieces, and the quality of the traditional perilla leaf formula particles in the market is uneven, such as authenticity is difficult to distinguish and quality is difficult to distinguish. Therefore, effective quality control of the particles of the pharmaceutical formulation has been sought after.
Perilla frutescens (Perilla frutescens (L.) Britton) is an annual upright herb plant of Tasmanian of Labiatae, and has the main part of stem leaf and fruit, and the leaf is an agent for inducing sweat, relieving cough, invigorating stomach and promoting urination with aromatic effect, and has analgesic, tranquilizing, and toxic substance removing effects, and can be used for treating common cold, abdominal pain and emesis due to fish and crab poisoning; the peduncles have the effects of calming the atmosphere and preventing miscarriage; zi can relieve cough, dispel phlegm, relieve dyspnea and dispel mental depression, and belongs to the pungent and warm exterior-releasing herbs of traditional Chinese medicine. The quality control method of the perilla leaf formula particles is not reported in the literature at present. In the chinese patent No. CN200310122427.0, volatile oil extracted from perilla leaves in a compound preparation is used as a characteristic of a utilization index, and in the chinese patent No. CN201410242575.4, volatile oil extracted from perilla leaves in a compound preparation is used as a qualitative identification index, however, the quality control index number of the above method is relatively single, and qualitative and quantitative quality detection cannot be performed comprehensively and accurately.
In summary, the prior art lacks a method capable of comprehensively and accurately controlling the quality of the perilla leaf formula particles so as to ensure the high quality of the perilla leaf formula particles and the high qualification rate in the production process.
Disclosure of Invention
In order to solve the problem that the prior art lacks a method capable of comprehensively and accurately controlling the quality of the perilla leaf formula particles, and also to ensure the high quality of the perilla leaf formula particles and the high qualification rate in the production process, the first aspect of the invention provides a whole process quality detection method in the preparation of the perilla leaf formula particles, which comprises a quality detection method of S1 perilla leaf decoction pieces, a quality detection method of S2 perilla leaf intermediates and a quality detection method of S3 perilla leaf formula particles;
in the quality detection of S1, S2 and S3, scutellarin and rosmarinic acid are used as characteristic indexes, and volatile oil, scutellarin and rosmarinic acid are used as content indexes for detection;
s1, a quality detection method of perilla leaf decoction pieces comprises the following steps:
and (3) detecting characteristic indexes: the method comprises the steps of taking a scutellarin solution, a rosmarinic acid solution and an elemene solution as reference substance solutions, taking a solution obtained by decocting a perilla leaf reference medicinal material as a reference substance solution of the reference medicinal material, taking a solution of perilla leaf decoction piece powder as a sample solution, and measuring by high performance liquid chromatography; analyzing and comparing the characteristic patterns of the reference substance solution and the sample solution, wherein the characteristic patterns of the sample solution should show 5 characteristic peaks and correspond to the retention time of 5 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarin reference peak, the characteristic peak 5 should correspond to the retention time of the elemene reference peak, the characteristic peak corresponding to the rosmarin reference is the S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within + -10% of the specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.44;
And (3) content index detection: the volatile oil content of the perilla leaf decoction pieces is not less than 0.40% (ml/g) measured by volatile oil measurement method; the method comprises the steps of taking a scutellarin solution and a rosmarinic acid solution as reference substance solutions, taking a solution of perilla leaf decoction piece powder as a test substance solution, and determining that the mass percent of scutellarin in the perilla leaf decoction pieces is not less than 0.08% and the mass percent of rosmarinic acid is not less than 0.36% by high performance liquid chromatography;
s2, a quality detection method of a perilla leaf intermediate comprises the following steps:
and (3) detecting characteristic indexes: the method comprises the steps of taking a scutellarin solution and a rosmarinic acid solution as reference substance solutions of reference substances, taking a solution obtained by decocting a perilla leaf reference medicinal material as the reference substance solution of the reference medicinal material, taking a solution of a perilla leaf intermediate as a sample solution, and measuring by high performance liquid chromatography; analyzing and comparing the characteristic patterns of the reference substance solution and the sample solution, wherein the characteristic patterns of the sample solution should show 4 characteristic peaks and correspond to the retention time of the 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the characteristic peak of the scutellarin reference substance, the characteristic peak 4 should correspond to the retention time of the reference substance peak of the rosmarinic acid reference substance, the characteristic peak corresponding to the rosmarinic acid reference substance is taken as an S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.45;
And (3) content index detection: the volatile oil content of the perilla leaf intermediate measured by the volatile oil measuring method is 0.18% -0.35% (ml/g); the method comprises the steps of taking a scutellarin solution and a rosmarinic acid solution as reference substance solutions, taking a solution of perilla leaf decoction piece powder as a sample solution, and measuring the mass percent of scutellarin in a perilla leaf intermediate by a high performance liquid chromatography to be 0.18-1.00%, wherein the mass percent of rosmarinic acid is 0.18-2.00%;
s3, a quality detection method of perilla leaf formula particles comprises the following steps:
the feature index detection is the same as S2;
and (3) content index detection: the volatile oil content of the perilla leaf intermediate measured by the volatile oil measuring method is 0.18% -0.35% (ml/g); the scutellarin solution and the rosmarinic acid solution are used as reference substance solutions, the solution of the perilla leaf decoction piece powder is used as a test substance solution, and the content of the scutellarin in each 1g of the perilla leaf formula particles is 1.5-10.0 mg and the content of the rosmarinic acid in each 1g of the perilla leaf formula particles is 1.5-20.0 mg measured by a high performance liquid chromatography.
The invention relates to a whole process quality detection method in preparation of perilla leaf formula particles, which specifically comprises the following steps:
s1: the quality detection method of the perilla leaf decoction pieces comprises the following steps:
S1.1: authentication, the authentication comprising the steps of:
s1.1.1: the characteristic identification of the perilla leaves is carried out according to Chinese pharmacopoeia: the leaves of the perilla leaf decoction pieces are crushed by multi-shrinkage and curling, the leaves are flattened into an oval shape with the length of 4-11 cm and the width of 2.5-9 cm, the front ends are long or sharp, the base is round or wide and wedge-shaped, the edges are round and saw-toothed, the two sides are purple or the upper surface is green, the lower surface is purple, grey white hair is thinned, the lower surface is provided with a plurality of concave punctiform gland scales, the leaf stalks are 2-7 cm long, purple or purple green, the quality is crisp, the diameter of the branches is 2-5 mm for the people with tender branches, the purple green is provided with marrow, faint scent and slight pungent taste in the middle part of the section;
s1.1.2: carrying out leaf surface flaking microscopic identification on perilla leaves according to Chinese pharmacopoeia: some cells in the epidermis contain purple pigment, and 10% hydrochloric acid solution is dripped to immediately display red; or dropwise adding 5% potassium hydroxide solution to obtain bright green color, and turning into yellow green color; the perilla leaf decoction piece powder is brown green, is not 1-7 cells of glandular hair, the diameter is 16-346 mu m, the surface is provided with linear textures, some cells are full of mauve or pink matters, the glandular hair is 2 cells, the diameter is 17-36 mu m, stem single cells, glandular scales are always broken, the head is 4-8 cells, upper and lower epidermis cells are irregular, the vertical peripheral wall is wavy and bent, the air holes are straight-shaft, the lower epidermis air holes are more, calcium oxalate cluster crystals are tiny, and the calcium oxalate cluster crystals exist in mesophyll cells;
S1.1.3: taking a perillaldehyde solution as a reference substance solution, taking a volatile oil solution extracted from folium Perillae decoction pieces as a sample solution, and measuring according to a thin layer chromatography in Chinese pharmacopoeia; analyzing and comparing chromatograms of the reference substance solution and the sample solution, wherein the sample chromatogram should be at the position corresponding to the reference substance chromatogram and show spots with the same color;
s1.1.4: taking folium Perillae control medicinal material solution as control solution, taking folium Perillae decoction piece powder solution as test solution, and determining by thin layer chromatography in Chinese pharmacopoeia; analyzing and comparing chromatograms of the reference substance solution and the sample solution, wherein the sample chromatogram should be at the position corresponding to the reference substance chromatogram and show spots with the same color;
s1.1.5 characteristic index detection: the method comprises the steps of taking a scutellarin solution, a rosmarinic acid solution and an elemene solution as reference substance solutions, taking a solution obtained by decocting a perilla leaf reference medicinal material as a reference substance solution, taking a solution of perilla leaf decoction piece powder as a sample solution, and measuring by high performance liquid chromatography; analyzing and comparing the characteristic patterns of the reference substance solution and the sample solution, wherein the characteristic patterns of the sample solution should show 5 characteristic peaks and correspond to the retention time of 5 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarin reference peak, the characteristic peak 5 should correspond to the retention time of the elemene reference peak, the characteristic peak corresponding to the rosmarin reference is the S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within + -10% of the specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.44;
S1.2: and (3) content index detection:
s1.2.1: measuring the content of volatile oil extracted from folium Perillae decoction pieces according to volatile oil measurement method in Chinese pharmacopoeia; analyzing the content measurement result of the volatile oil, wherein the content of the volatile oil contained in the perilla leaf decoction pieces is not less than 0.40% (ml/g); and
s1.2.2: taking scutellarin solution and rosmarinic acid solution as reference substance solutions, taking solution of folium Perillae decoction piece powder as sample solution, and determining by high performance liquid chromatography; analyzing and measuring the content of scutellarin and rosmarinic acid contained in the perilla leaf decoction pieces, wherein the mass percent of scutellarin in the perilla leaf decoction pieces is not less than 0.08% and the mass percent of rosmarinic acid is not less than 0.36% according to the dry product;
s2: the quality detection method of the perilla leaf intermediate comprises the following steps:
s2.1: authentication, the authentication comprising the steps of:
s2.1.1 trait identification: the purple perilla leaf intermediate is yellow-brown to tan powder, has faint scent and slightly bitter taste;
s2.1.2: as well as S1.1.3;
s2.1.3: as well as S1.1.4;
s2.1.4: and (3) detecting characteristic indexes: the method comprises the steps of taking a scutellarin solution and a rosmarinic acid solution as reference substance solutions of reference substances, taking a solution obtained by decocting a perilla leaf reference medicinal material as the reference substance solution of the reference medicinal material, taking a solution of a perilla leaf intermediate as a sample solution, and measuring by high performance liquid chromatography; analyzing and comparing the characteristic patterns of the reference substance solution and the sample solution, wherein the characteristic patterns of the sample solution should show 4 characteristic peaks and correspond to the retention time of the 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the characteristic peak of the scutellarin reference substance, the characteristic peak 4 should correspond to the retention time of the reference substance peak of the rosmarinic acid reference substance, the characteristic peak corresponding to the rosmarinic acid reference substance is taken as an S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.45;
S2.2: and (3) content index detection:
s2.2.1: the steps are the same as S1.2.1, except that the content of volatile oil contained in the intermediate of the perilla leaf is 0.18% -0.35% (ml/g); and
s2.2.2: the steps are S1.2.2, wherein the difference is that the mass percent of scutellarin in the purple perilla leaf intermediate is 0.18-1.00% and the mass percent of rosmarinic acid is 0.18-2.00% based on the dry product;
s3: the quality detection method of the perilla leaf formula granule comprises the following steps:
s3.1: authentication, the authentication comprising the steps of:
s3.1.1 trait identification: the perilla leaf formula particles are yellowish brown to tan particles, have faint scent and slightly bitter taste;
s3.1.2: as well as S1.1.3;
s3.1.3: as well as S1.1.4;
s3.1.4: the characteristic index is detected as S2.1.4;
s3.2: and (3) content index detection:
s3.2.1: the difference with S1.2.1 is that the volatile oil content contained in the perilla leaf formula granule is 0.18% -0.35% (ml/g); and
s3.2.2: the difference is that the scutellarin content in each 1g of the perilla leaf formula granule is 1.5-10.0 mg, and the rosmarinic acid content in each 1g of the perilla leaf formula granule is 1.5-20.0 mg.
Further, the step S1 in the quality detection method of the perilla leaf decoction pieces further comprises the steps of: s0: the quality detection method of the perilla leaf medicinal materials is carried out according to the quality detection method under the item of perilla leaf in Chinese pharmacopoeia.
Further, step S1.1.5 includes:
preparation of reference solution: taking appropriate amounts of scutellarin reference substance solution, rosmarinic acid reference substance solution, elemene reference substance solution and perilla leaf reference substance solution, precisely weighing, and adding 70% methanol to obtain solutions containing 50 μg, 80 μg and 90 μg respectively per 1 ml;
preparation of test solution: taking about 0.3g of perilla leaf decoction piece powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate;
high performance liquid chromatography, as shown in the preferred examples: octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.6 mu m; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid solution is taken as a mobile phase B; gradient elution is carried out for 0-4 min,17% of A and 83% of B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm; the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of reference substance solution of reference substance, 1 μl of reference substance solution of folium Perillae reference medicinal material and 1 μl of sample solution respectively, and measuring with liquid chromatograph;
Analysis: the characteristic spectrum of the sample solution should show 5 characteristic peaks and correspond to 5 characteristic peak retention times in the chromatogram of the reference substance of the reference medicinal material; wherein, the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarinic acid reference peak, and the characteristic peak 5 should correspond to the retention time of the elemene reference peak. The characteristic peak corresponding to the rosmarinic acid reference substance is S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of the specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.44.
Further, step S1.2.2 includes:
preparation of a control solution: taking scutellarin as a reference substance and a right amount of rosmarinic acid as a reference substance, precisely weighing, adding 70% methanol to prepare mixed solutions containing 50 mug and 80 mug respectively per 1ml, and shaking uniformly to obtain the scutellarin;
preparation of test solution: taking 0.3g of perilla leaf decoction piece powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, and taking the subsequent filtrate to obtain the perilla leaf decoction piece powder;
High performance liquid chromatography, as shown in the preferred examples: octadecylsilane chemically bonded silica is used as a filling agent of a chromatographic column; acetonitrile solution is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0-4 min, the time is 17 percent A, and the time is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the detection wavelength is 330nm; column temperature 40 ℃; the flow rate is 0.3ml per minute, and the theoretical plate number is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement to obtain scutellarin and rosmarinic acid contents;
analysis: and analyzing and comparing the measurement results of the test solution and the reference solution, wherein the mass percent of scutellarin in the perilla leaf decoction pieces is not less than 0.08% and the mass percent of rosmarinic acid is not less than 0.36% according to the dry product.
Further, step S2.1.4 includes:
Preparation of reference solution: taking appropriate amounts of scutellarin reference substance solution and rosmarinic acid reference substance solution, precisely weighing, and respectively adding methanol to obtain 50 μg/lml scutellarin-containing solution and 80 μg/rosmarinic acid-containing solution; taking 0.3g of perilla leaf reference medicinal material powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of water, sealing, weighing, heating and decocting for 30 minutes, cooling, weighing again, supplementing the reduced weight with water, shaking uniformly, filtering, and collecting the subsequent filtrate;
preparation of test solution: taking 0.1g of perilla leaf intermediate, precisely weighing, placing into a conical flask with a plug, precisely adding 20ml of 70% methanol, weighing, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the perilla leaf intermediate;
high performance liquid chromatography, as shown in the preferred examples: octadecylsilane chemically bonded silica is used as a filler (column length is 150mm, inner diameter is 2.1mm, and particle diameter is 1.6 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0 to 4min, the concentration of the phosphoric acid solution is 17 percent A, and the concentration of the phosphoric acid solution is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm, and the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peaks; precisely sucking 1 μl of each of the reference solution and the sample solution, and measuring with a liquid chromatograph;
Analysis: the characteristic spectrum of the sample solution should show 4 characteristic peaks and correspond to the retention time of 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarinic acid reference peak, the characteristic peak corresponding to the rosmarinic acid reference peak is taken as an S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.45.
Further, step S1.1.3 includes: adding n-hexane into volatile oil extracted from folium Perillae decoction pieces to obtain sample solution containing 10 μl per 1 ml; taking perillaldehyde reference substance, and adding n-hexane to prepare reference substance solution containing 10 μl per 1 ml; according to the thin layer chromatography determination in Chinese pharmacopoeia, sucking 2 μl of each of the sample solution and the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with n-hexane-ethyl acetate (15:1) as developing agent, taking out, air drying, and spraying dinitrophenylhydrazine ethanol solution; and analyzing and comparing the measurement results of the test solution and the reference solution, wherein the test solution and the reference solution are subjected to the analysis, and spots with the same color are displayed on the positions corresponding to the chromatogram of the reference solution.
Further, step S1.1.4 includes: adding 25ml of methanol into 0.5g of folium Perillae decoction pieces, performing ultrasonic treatment for 30 min, filtering, concentrating the filtrate to dryness, and adding 2ml of methanol to dissolve to obtain sample solution; another 0.5g of perilla leaf reference medicinal material is prepared into a reference medicinal material solution by the same method; according to thin layer chromatography test in Chinese pharmacopoeia, sucking 3 μl of each of the test solution and the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-formic acid-water (9:0.5:1:0.5) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, inspecting under 365nm ultraviolet lamp, and analyzing and comparing the measurement results of the test solution and the reference solution, wherein the test solution should have fluorescent spots with the same color at the positions corresponding to the reference solution.
Further, step s1.2.1 includes: and (3) determining the content of volatile oil in the perilla leaf decoction pieces according to a volatile oil determination method in Chinese pharmacopoeia, keeping micro-boiling for 2.5 hours, and analyzing the determination result, wherein the content of volatile oil in the perilla leaf decoction pieces is not less than 0.40% (ml/g).
The second aspect of the application provides a perilla leaf formula particle, which is prepared by adopting the whole process quality detection method.
The third aspect of the application provides a preparation method of perilla leaf formula particles by adopting the whole process quality detection method.
Preferably, the "chinese pharmacopoeia" is not limited to a specific version.
For example, the above-mentioned "chinese pharmacopoeia" all refer to the 2020 edition.
After the technical scheme is adopted, compared with the prior art, the method has the following beneficial effects:
1. the application provides a plurality of characteristic indexes and content indexes in the quality detection process of perilla leaf decoction pieces, she Zhongjian body and perilla leaf formula granules for the first time, thereby improving the accuracy of true and false identification and quality evaluation.
2. The technical scheme of the application defines a novel whole process quality detection method in the preparation process of the perilla leaf formula particles, and the method comprises a quality detection method of perilla leaf decoction pieces, a quality detection method of perilla leaf intermediates and a quality detection method of the perilla leaf formula particles, has strong specificity and good reproducibility, and can effectively control the quality of each link in the preparation process of the perilla leaf formula particles; the whole process quality detection method can ensure the authenticity and high qualification rate of the perilla leaf decoction pieces, the perilla She Zhongjian body and the perilla leaf formula particles, and can ensure that the difference between the perilla She Zhongjian body and the perilla leaf formula particles among different production batches is small (namely, the stability is high); and the uniformity of the perilla She Zhongjian body and the perilla leaf formula granules in the same production batch is high.
3. The whole process quality detection method further optimizes the preparation process of the perilla leaf decoction pieces, the perilla She Zhongjian body and the perilla leaf formula particles.
Drawings
FIG. 1 is a comparative characteristic spectrum for identifying decoction pieces of folium Perillae, wherein the figure has 5 characteristic peaks;
FIG. 2 is a characteristic map of a control medicinal material for identifying the intermediate of perilla leaves, wherein the characteristic map has 4 characteristic peaks in total;
FIG. 3 is a control profile identifying the intermediate of Perilla leaf, showing a total of 4 characteristic peaks;
FIG. 4 is a graph of a control characteristic spectrum identifying perilla leaf formula particles, wherein the graph has 4 characteristic peaks in total;
in fig. 5, a is a thin layer identification spectrum of a perilla leaf intermediate and a perilla leaf reference medicine in different batches, and B is a thin layer identification spectrum of volatile oil and a perilla aldehyde reference medicine in different batches of the perilla leaf intermediate;
FIG. 6 is a characteristic spectrum of the intermediates of the perilla leaves of different batches, and a spectrum of the control medicinal materials of the perilla leaves and the reference substances (scutellarin and rosmarinic acid) of the control substances;
FIG. 7 is a graph showing characteristic patterns of different batches of leaf intermediates of Perilla frutescens and patterns of reference substances (scutellarin and rosmarinic acid);
in fig. 8, a is a thin layer identification spectrum of perilla leaf formula particles of different batches and a perilla leaf control medicinal material, and B is a thin layer identification spectrum of volatile oil of the perilla leaf formula particles of different batches and a perilla aldehyde control;
FIG. 9 is a characteristic spectrum of different batches of perilla leaf formula granules, and a spectrum of a perilla leaf control medicinal material and a reference substance (scutellarin and rosmarinic acid) of the control;
fig. 10 shows characteristic patterns of different batches of perilla leaf formula granules and patterns of reference substances (scutellarin and rosmarinic acid).
Detailed Description
Advantages of the invention are further illustrated in the following description, taken in conjunction with the accompanying drawings and detailed description. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
1. Examples: preparation of perilla leaf formula granule and total process quality control
Step 1: processing dried leaves (or twigs) of Perilla Perilla frutescens (L.) Britt of Labiatae to obtain folium Perillae decoction pieces (removing impurities and old stems), decocting 4000g of folium Perillae decoction pieces with water, and collecting appropriate amount of volatile oil;
step 2: filtering the liquid decocted in the step 1 to obtain filtrate, and concentrating the filtrate into clear paste (the paste yield of the dry extract is 14% -25%);
step 3: adding the volatile oil clathrate into the fluid extract, drying, adding appropriate amount of adjuvant, mixing, granulating, and making into 1000g folium Perillae granule.
Preferably, in step 1, the quality detection method of the perilla leaf decoction pieces comprises the following steps:
S1.1: authentication, the authentication comprising the steps of:
s1.1.1: character identification: the leaves of the product are crimped and curled, crushed and flattened into an oval shape with the length of 4-11 cm and the width of 2.5-9 cm. The tip is long or sharp, the base is round or wide wedge-shaped, and the edge is provided with round saw teeth. Purple on both sides or green on the upper surface, purple on the lower surface, grey white hair on the lower surface, and a plurality of concave punctiform glandular scales on the lower surface. The length of the petiole is 2-7 cm, purple or purple green. It is crisp. With tender branches, the diameter of the branches is 2-5 mm, the branches are purple green, and the middle part of the cross section is provided with marrow. Fragrant smell, slightly pungent taste;
s1.1.2: leaf surface slide microscopic identification: some cells in the epidermis contain purple pigment, and 10% hydrochloric acid solution is dripped to immediately display red; or dropwise adding 5% potassium hydroxide solution to obtain bright green color, and turning into yellow green color; the powder is brown and green, the diameter of the powder is between 1 and 7 cells, the diameter of the powder is between 16 and 346 mu m, the surface of the powder is provided with linear textures, some cells are full of mauve or pink matters, the glandular hair is mostly 2 cells, the diameter of the powder is between 17 and 36 mu m, single stem cells are always broken, the glandular scales are always broken, the head is 4 to 8 cells, the upper and lower epidermis cells are irregularly shaped, the vertical peripheral wall is wavy and bent, the air holes are straight-shaft, the air holes of the lower epidermis are more, calcium oxalate cluster crystals are tiny, and the calcium oxalate cluster crystals exist in mesophyll cells;
S1.1.3: adding n-hexane into volatile oil extracted from folium Perillae decoction pieces to obtain sample solution containing 10 μl per 1 ml; taking perillaldehyde reference substance, and adding n-hexane to prepare reference substance solution containing 10 μl per 1 ml; according to the measurement of four general rules 0502 in China pharmacopoeia 2020, 2 μl of each of the test sample solution and the reference sample solution is absorbed and respectively spotted on the same silica gel G thin layer plate, and the mixture is spread with n-hexane-ethyl acetate (15:1) as a developing agent, taken out, dried and sprayed with dinitrophenylhydrazine ethanol test solution; analyzing and comparing the measurement results of the test sample solution and the reference sample solution, wherein the chromatogram of the test sample is at the position corresponding to the chromatogram of the reference sample and shows spots with the same color;
s1.1.4: adding 25ml of methanol into 0.5g of folium Perillae decoction pieces, performing ultrasonic treatment for 30 min, filtering, concentrating the filtrate to dryness, and adding 2ml of methanol to dissolve to obtain sample solution; another 0.5g of perilla leaf reference medicinal material is prepared into a reference medicinal material solution by the same method; according to the four general rules 0502 thin layer chromatography test of Chinese pharmacopoeia 2020, sucking 3 μl of each of the test solution and the reference solution, respectively placing on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-formic acid-water (9:0.5:1:0.5) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of the spot becomes clear, inspecting under 365nm ultraviolet lamp, analyzing and comparing the measurement results of the test solution and the reference solution, wherein the test chromatograph should display fluorescent spots of the same color at the position corresponding to the reference chromatograph;
S1.1.5 preparation of reference solution: taking appropriate amounts of scutellarin reference substance solution, rosmarinic acid reference substance solution, elemene reference substance solution and perilla leaf reference substance solution, precisely weighing, and adding 70% methanol to obtain solutions containing 50 μg, 80 μg and 90 μg respectively per 1 ml;
preparation of test solution: taking about 0.3g of perilla leaf decoction piece powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate;
the method is measured according to the high performance liquid chromatography of the four-part general rule 0512 in the Chinese pharmacopoeia 2020 edition.
The method for measuring high performance liquid chromatography according to the present invention is preferably as follows: octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.6 mu m; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid solution is taken as a mobile phase B; gradient elution is carried out for 0-4 min,17% of A and 83% of B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm; the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of reference substance solution of reference substance, 1 μl of reference substance solution of folium Perillae reference medicinal material and 1 μl of sample solution respectively, and measuring with liquid chromatograph;
Analysis: the characteristic spectrum of the sample solution should show 5 characteristic peaks and correspond to 5 characteristic peak retention times in the chromatogram of the reference substance of the reference medicinal material; wherein, the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarinic acid reference peak, and the characteristic peak 5 should correspond to the retention time of the elemene reference peak. Calculating the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak, wherein the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.44; the contrast characteristic map of the perilla leaf decoction pieces is shown in figure 1;
s1.2: content determination, comprising the steps of:
s1.2.1: determining the volatile oil content in the perilla leaf decoction pieces according to the general rule 2204 volatile oil determination method of the four parts of the edition of Chinese pharmacopoeia 2020, keeping micro-boiling for 2.5 hours, and analyzing the determination result, wherein the volatile oil content in the perilla leaf decoction pieces is not less than 0.40% (ml/g); and
s1.2.2: preparation of a control solution: taking scutellarin as a reference substance and a right amount of rosmarinic acid as a reference substance, precisely weighing, adding 70% methanol to prepare mixed solutions containing 50 mug and 80 mug respectively per 1ml, and shaking uniformly to obtain the scutellarin;
Preparation of test solution: taking 0.3g of perilla leaf decoction piece powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, and taking the subsequent filtrate to obtain the perilla leaf decoction piece powder;
the method is measured according to the high performance liquid chromatography of the four-part general rule 0512 in the Chinese pharmacopoeia 2020 edition.
The method for measuring high performance liquid chromatography according to the present invention is preferably as follows: octadecylsilane chemically bonded silica is used as a filling agent of a chromatographic column; acetonitrile solution is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0-4 min, the time is 17 percent A, and the time is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the detection wavelength is 330nm; column temperature 40 ℃; the flow rate is 0.3ml per minute, and the theoretical plate number is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement to obtain scutellarin and rosmarinic acid contents;
Analysis: analyzing and comparing the measurement results of the test solution and the reference solution, wherein the weight percentage of scutellarin in the perilla leaf decoction pieces is not less than 0.08% and the weight percentage of rosmarinic acid is not less than 0.36% based on the dry product;
s1.2.3 inspection: the content of the water measured by a fourth method of the four-part rule 0832 water measuring method of the Chinese pharmacopoeia 2020 edition is not more than 12.0 percent; the sulfur dioxide residue measured by the sulfur dioxide residue measuring method of 2331 of the fourth edition of the Chinese pharmacopoeia 2020 edition is not more than 150mg/kg; according to the four-part general rule 0412 inductively coupled plasma mass spectrometry of Chinese pharmacopoeia 2020, the content of heavy metals and harmful elements is measured, lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20mg/kg.
Preferably, the perilla leaf decoction pieces obtained according to the quality detection method of the above perilla leaf decoction pieces are used in the above step 2.
Preferably, in step 2, the method for detecting the quality of the perilla She Zhongjian body (i.e., dry extract) comprises the following steps:
s2.1: authentication, the authentication comprising the steps of:
s2.1.1 trait identification: the purple perilla leaf intermediate is yellow-brown to tan powder, has faint scent and slightly bitter taste;
S2.1.2: adding n-hexane into volatile oil extracted from folium Perillae intermediate to obtain sample solution containing 10 μl per 1 ml; taking perillaldehyde reference substance, and adding n-hexane to prepare reference substance solution containing 10 μl per 1 ml; according to the measurement of four general rules 0502 in China pharmacopoeia 2020, 2 μl of each of the test sample solution and the reference sample solution is absorbed and respectively spotted on the same silica gel G thin layer plate, and the mixture is spread with n-hexane-ethyl acetate (15:1) as a developing agent, taken out, dried and sprayed with dinitrophenylhydrazine ethanol test solution; analyzing and comparing the measurement results of the test sample solution and the reference sample solution, wherein the chromatogram of the test sample is at the position corresponding to the chromatogram of the reference sample and shows spots with the same color;
s2.1.3: adding 25ml of methanol into 0.5g of perilla leaf intermediate, carrying out ultrasonic treatment for 30 minutes, filtering, concentrating the filtrate to dryness, and adding 2ml of methanol to dissolve the filtrate to obtain a sample solution; another 0.5g of perilla leaf reference medicinal material is prepared into a reference medicinal material solution by the same method; according to the four general rules 0502 thin layer chromatography test of Chinese pharmacopoeia 2020, sucking 3 μl of each of the test solution and the reference solution, respectively placing on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-formic acid-water (9:0.5:1:0.5) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of the spot becomes clear, inspecting under 365nm ultraviolet lamp, analyzing and comparing the measurement results of the test solution and the reference solution, wherein the test chromatograph should display fluorescent spots of the same color at the position corresponding to the reference chromatograph;
S2.1.4: preparation of reference solution: taking appropriate amounts of scutellarin reference substance solution and rosmarinic acid reference substance solution, precisely weighing, and respectively adding methanol to obtain 50 μg/lml scutellarin-containing solution and 80 μg/rosmarinic acid-containing solution; taking 0.3g of perilla leaf reference medicinal material powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of water, sealing, weighing, heating and decocting for 30 minutes, cooling, weighing again, supplementing the reduced weight with water, shaking uniformly, filtering, and collecting the subsequent filtrate;
preparation of test solution: taking 0.1g of perilla leaf intermediate, precisely weighing, placing into a conical flask with a plug, precisely adding 20ml of 70% methanol, weighing, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the perilla leaf intermediate;
and (3) measuring: the method is measured according to the high performance liquid chromatography of the four-part general rule 0512 in the Chinese pharmacopoeia 2020 edition.
The method for measuring high performance liquid chromatography according to the present invention is preferably as follows: octadecylsilane chemically bonded silica is used as a filler (column length is 150mm, inner diameter is 2.1mm, and particle diameter is 1.6 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0 to 4min, the concentration of the phosphoric acid solution is 17 percent A, and the concentration of the phosphoric acid solution is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm, and the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peaks; precisely sucking 1 μl of each of the reference solution and the sample solution, and measuring with a liquid chromatograph;
Analysis: the characteristic spectrum of the sample solution should show 4 characteristic peaks and correspond to the retention time of 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarinic acid reference peak, the characteristic peak corresponding to the rosmarinic acid reference is taken as an S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.45; the characteristic spectrum of the contrast medicinal material of the intermediate of the perilla leaf is shown in figure 2, and the characteristic spectrum of the contrast medicinal material of the intermediate of the perilla leaf is shown in figure 3;
s2.2: content determination, comprising the steps of:
s2.2.1: determining the content of volatile oil in the perilla leaf intermediate according to the general rule 2204 volatile oil determination method of the four parts of the edition of Chinese pharmacopoeia 2020, keeping micro-boiling for 2.5 hours, and analyzing the determination result, wherein the content of the volatile oil in the perilla leaf intermediate is 0.18% -0.35% (ml/g); and
s2.2.2: preparation of a control solution: taking scutellarin as a reference substance and a right amount of rosmarinic acid as a reference substance, precisely weighing, adding 70% methanol to prepare mixed solutions containing 50 mug and 80 mug respectively per 1ml, and shaking uniformly to obtain the scutellarin;
Preparation of test solution: precisely weighing 0.3g of perilla leaf intermediate, placing into a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, and collecting the subsequent filtrate;
the method is measured according to the high performance liquid chromatography of the four-part general rule 0512 in the Chinese pharmacopoeia 2020 edition.
The method for measuring high performance liquid chromatography of the present invention is further preferably: octadecylsilane chemically bonded silica is used as a filling agent of a chromatographic column; acetonitrile solution is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0-4 min, the time is 17 percent A, and the time is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the detection wavelength is 330nm; column temperature 40 ℃; the flow rate is 0.3ml per minute, and the theoretical plate number is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement to obtain scutellarin and rosmarinic acid contents;
Analysis: analyzing and comparing the measurement results of the test solution and the reference solution, wherein the mass percent of scutellarin in the perilla leaf intermediate is 0.18-1.00% and the mass percent of rosmarinic acid is 0.18-2.00% based on the dry product;
s2.2.3 inspection: the moisture, drying weight loss, solubility, loading and microorganism limit of the purple perilla leaf intermediate are regulated according to various regulations under the four general rules 0104 granule item of the Chinese pharmacopoeia 2020 edition.
Preferably, a fluid extract (dry extract) obtained in conformity with the quality detection method of the above-mentioned perilla leaf intermediate is used in the step 3.
Preferably, in step 3, the quality detection method of the perilla leaf formula particle comprises the following steps:
s3.1: authentication, the authentication comprising the steps of:
s3.1.1 trait identification: the perilla leaf formula particles are yellowish brown to tan particles, have faint scent and slightly bitter taste;
s3.1.2: adding n-hexane into volatile oil extracted from folium Perillae decoction pieces to obtain sample solution containing 10 μl per 1 ml; taking perillaldehyde reference substance, and adding n-hexane to prepare reference substance solution containing 10 μl per 1 ml; according to the measurement of four general rules 0502 in China pharmacopoeia 2020, 2 μl of each of the test sample solution and the reference sample solution is absorbed and respectively spotted on the same silica gel G thin layer plate, and the mixture is spread with n-hexane-ethyl acetate (15:1) as a developing agent, taken out, dried and sprayed with dinitrophenylhydrazine ethanol test solution; analyzing and comparing the measurement results of the test sample solution and the reference sample solution, wherein the chromatogram of the test sample is at the position corresponding to the chromatogram of the reference sample and shows spots with the same color;
S3.1.3: adding 25ml of methanol into 0.5g of folium Perillae decoction pieces, performing ultrasonic treatment for 30 min, filtering, concentrating the filtrate to dryness, and adding 2ml of methanol to dissolve to obtain sample solution; another 0.5g of perilla leaf reference medicinal material is prepared into a reference medicinal material solution by the same method; according to the four general rules 0502 thin layer chromatography test of Chinese pharmacopoeia 2020, sucking 3 μl of each of the test solution and the reference solution, respectively placing on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-formic acid-water (9:0.5:1:0.5) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of the spot becomes clear, inspecting under 365nm ultraviolet lamp, analyzing and comparing the measurement results of the test solution and the reference solution, wherein the test chromatograph should display fluorescent spots of the same color at the position corresponding to the reference chromatograph;
s3.1.4: preparation of reference solution: taking appropriate amounts of scutellarin reference substance solution and rosmarinic acid reference substance solution, precisely weighing, and respectively adding methanol to obtain 50 μg/lml scutellarin-containing solution and 80 μg/rosmarinic acid-containing solution; taking 0.3g of perilla leaf reference medicinal material powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of water, sealing, weighing, heating and decocting for 30 minutes, cooling, weighing again, supplementing the reduced weight with water, shaking uniformly, filtering, and collecting the subsequent filtrate;
Preparation of test solution: taking 0.1g of perilla leaf intermediate, precisely weighing, placing into a conical flask with a plug, precisely adding 20ml of 70% methanol, weighing, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the perilla leaf intermediate;
and (3) measuring: the method is measured according to the high performance liquid chromatography of the four-part general rule 0512 in the Chinese pharmacopoeia 2020 edition.
The method for measuring high performance liquid chromatography of the present invention is further preferably: octadecylsilane chemically bonded silica is used as a filler (column length is 150mm, inner diameter is 2.1mm, and particle diameter is 1.6 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0 to 4min, the concentration of the phosphoric acid solution is 17 percent A, and the concentration of the phosphoric acid solution is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm, and the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peaks; precisely sucking 1 μl of each of the reference solution and the sample solution, and measuring with high performance liquid chromatograph;
Analysis: the characteristic spectrum of the sample solution should show 4 characteristic peaks and correspond to the retention time of 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarinic acid reference peak, the characteristic peak corresponding to the rosmarinic acid reference is taken as an S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.45; the comparison characteristic map of the perilla leaf formula granule is shown in figure 4;
s3.2: content determination, comprising the steps of:
s3.2.1: determining the volatile oil content in the perilla leaf decoction pieces according to the general rule 2204 volatile oil determination method of the four parts of the edition of Chinese pharmacopoeia 2020, keeping micro-boiling for 2.5 hours, and analyzing the determination result, wherein the volatile oil content in the perilla leaf formula particles is 0.18% -0.35% (ml/g); and
s3.2.2: preparation of a control solution: taking scutellarin as a reference substance and a right amount of rosmarinic acid as a reference substance, precisely weighing, adding 70% methanol to prepare mixed solutions containing 50 mug and 80 mug respectively per 1ml, and shaking uniformly to obtain the scutellarin;
Preparation of test solution: taking 0.3g of perilla leaf decoction piece powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, and taking the subsequent filtrate to obtain the perilla leaf decoction piece powder;
the method is measured according to the high performance liquid chromatography of the four-part general rule 0512 in the Chinese pharmacopoeia 2020 edition.
The method for measuring high performance liquid chromatography of the present invention is further preferably: octadecylsilane chemically bonded silica is used as a filling agent of a chromatographic column; acetonitrile solution is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0-4 min, the time is 17 percent A, and the time is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the detection wavelength is 330nm; column temperature 40 ℃; the flow rate is 0.3ml per minute, and the theoretical plate number is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement to obtain scutellarin and rosmarinic acid contents;
Analysis: analyzing and comparing the measurement results of the test solution and the reference solution, wherein the content of scutellarin in each 1g of the perilla leaf formula particles is 1.5-10.0 mg, and the content of rosmarinic acid in each 1g of the perilla leaf formula particles is 1.5-20.0 mg;
s3.2.3 inspection: the granularity, the moisture, the drying weight loss, the solubility, the loading difference, the loading and the microorganism limit of the perilla leaf formula granule are regulated according to the regulations under the four-part rule 0104 granule item in the Chinese pharmacopoeia 2020;
s3.2.4 extract content determination: the content of extract is measured according to the method for measuring the alcohol-soluble extract of 2201 in the fourth edition of the Chinese pharmacopoeia 2020, and the content of extract is not less than 12.0 percent by using ethanol as a solvent.
2. Effect examples
Stability tests were performed on different batches of perilla She Zhongjian body and perilla leaf formula particles prepared by the quality control method of the perilla leaf extract in the above examples, and homogeneity tests were performed on different samples of the perilla She Zhongjian body and the perilla leaf formula particles in the same batch.
(1) Stability test between different batches
1. Stability between different batches of Perilla leaf intermediate
1.1 thin layer authentication
1.1.1 methods of investigation
(1) Respectively taking 0.5g of each of batch 1, batch 2 and batch 3 of the perilla leaf intermediate, respectively adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 2ml of methanol into residues to dissolve the residues to obtain a sample solution. And adding 1g of perilla leaf reference medicinal material, adding 50ml of water, boiling for 30 minutes, filtering, evaporating filtrate to dryness, adding 25ml of methanol into residues, and preparing a reference medicinal material solution by the same method. Test by thin layer chromatography (rule 0502 of four parts of chinese pharmacopoeia 2020 edition), T:16 ℃, RH:50%, sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-formic acid-water (9:0.5:1:0.5) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, and inspecting under ultraviolet lamp (365 nm).
(2) Taking volatile oil of batch 1, batch 2 and batch 3 of folium Perillae intermediate respectively, and adding n-hexane to obtain solution containing 10 μl per 1ml, and making into test solution. And adding n-hexane into the perillaldehyde reference substance to prepare a solution with the concentration of 0 mu l per lml as the reference substance solution. Test by thin layer chromatography (rule 0502 of four parts of chinese pharmacopoeia 2020 edition), T:20 ℃, RH:60%, sucking 5-10 μl of the sample solution and 2 μl of the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with n-hexane-ethyl acetate (15:1) as developing agent, taking out, air drying, and spraying dinitrophenylhydrazine ethanol solution.
1.1.2 results of the study
The thin layer identification patterns of the perilla leaf intermediates and the perilla leaf control medicinal materials in different batches are shown as A in fig. 5, wherein 1 in A corresponds to batches 1 and 2, 3 corresponds to batches 2 and 3, and S corresponds to the perilla leaf control medicinal materials; the thin layer identification patterns of volatile oil of the intermediate of the perilla leaf and the perillaldehyde reference substance in different batches are shown as B in fig. 5, wherein 1 in B corresponds to batches 1 and 2, 3 corresponds to batches 2 and 3, and T corresponds to the perillaldehyde reference substance. As can be seen from a in fig. 5, in the sample chromatograms of the batch 1, the batch 2 and the batch 3 of the perilla leaf intermediate, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference medicinal material, and the stability is good without significant difference. As can be seen from fig. 5B, in the sample chromatograms of batch 1, batch 2 and batch 3 of the perilla leaf intermediate, spots of the same color appear at the positions corresponding to the chromatogram of the reference sample, and have no significant difference and good stability. From this, the difference between different batches of the perilla leaf intermediate prepared by the perilla leaf intermediate quality control method in the above embodiment is small and the stability is good.
1.2 characteristic Spectrum
1.2.1 methods of research
[ characteristic map ] is measured by high performance liquid chromatography (four-part rule 0512 in the year of the Chinese pharmacopoeia 2020).
[ chromatographic conditions and System applicability test ] octadecylsilane chemically bonded silica was used as a filler (column length: 150mm, inner diameter: 2.1mm, particle size: 1.6 μm, waters CORTECS T3 column); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to Table 1; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength was 215nm. The number of theoretical plates should be not less than 3000 calculated as rosmarinic acid peak.
TABLE 1 elution gradient for high performance liquid chromatography
Preparation of reference solution 0.3g of folium Perillae reference material is put into a conical flask with a plug, 25ml of water is added, the mixture is heated and decocted for 30 minutes, cooled, shaken well and filtered, and the subsequent filtrate is taken as reference solution of reference material. And (3) taking a proper amount of scutellarin reference substance and rosmarinic acid reference substance, precisely weighing, and respectively adding methanol to prepare solutions containing 50 mug of scutellarin and 80 mug of rosmarinic acid per lml as reference substance solutions of the reference substances.
Preparing test solution, namely taking 0.1g of each of batch 1 (comprising batch 1-1 and batch 1-2), batch 2 (comprising batch 2-1 and batch 2-2) and batch 3 (comprising batch 3-1 and batch 3-2) of the perilla leaf intermediate, precisely weighing, placing the materials into a conical flask with a plug, precisely adding 20ml of 70% methanol, weighing, performing ultrasonic treatment (with power of 250W and frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the loss weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
The measurement method comprises precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement to obtain the final product shown in Table 2.
The sample chromatogram should show 4 characteristic peaks and correspond to the retention time of 4 characteristic peaks in the control reference chromatogram; wherein peak 3 is scutellarin, peak 4 (S) is rosmarinic acid, and peak 3 and peak 4 should correspond to retention time of scutellarin and rosmarinic acid reference peak. The peak corresponding to the rosmarinic acid reference is S peak, and the relative retention time of the characteristic peaks 1, 2 and S peak is calculated, wherein the relative retention time is within + -10% of the specified value. The specified value is: 0.29 (Peak 1), 0.45 (Peak 2).
1.2.2 results of the study
The characteristic patterns of the intermediates of the perilla leaf, the patterns of the control medicinal materials of the perilla leaf and the reference substances (scutellarin and rosmarinic acid) of different batches are shown in fig. 6, 1-1 (4) in fig. 6 corresponds to batches 1-1, 1-2 (4) corresponds to batches 1-2, 2-1 (4) corresponds to batches 2-1, 2-2 (4) corresponds to batches 2-2, 3-1 (4) corresponds to batches 3-1, 3-2 (4) corresponds to batches 3-2, T (2) corresponds to the control substance of scutellarin and the reference substance of rosmarinic acid, and S (4) corresponds to the control medicinal materials of the perilla leaf.
As can be seen from table 2 and fig. 6, the characteristic patterns of the perilla leaf intermediates of lot 1, lot 2 and lot 3 each show 4 characteristic peaks, and correspond to the retention time of the 4 characteristic peaks in the chromatogram of the reference material. Wherein peak 3 and peak 4 correspond to the retention time of scutellarin and rosmarinic acid reference peak respectively. The relative retention time of peak 1 and peak 2 is within + -10% of the prescribed value.
The detailed data are as follows:
TABLE 2 relative retention times for batch 1, batch 2, batch 3 of Perilla leaf intermediates
| Batch of | Peak 1 | Peak 2 | Peak 3 | Peak 4 (S) |
| Specified value | 0.29±10％ | 0.45±10％ | Scutellarin (scutellarin) | Rosmarinic acid |
| 1-1 | 0.310 | 0.491 | / | 1.000 |
| 1-2 | 0.310 | 0.491 | / | 1.000 |
| 2-1 | 0.308 | 0.483 | / | 1.000 |
| 2-2 | 0.308 | 0.483 | / | 1.000 |
| 3-1 | 0.306 | 0.481 | / | 1.000 |
| 3-2 | 0.306 | 0.481 | / | 1.000 |
| RSD(％) | 0.58 | 0.98 | / | / |
1.3 content determination
1.3.1. Research method
[ MEANS FOR SOLVING ] volatile oil is measured by volatile oil assay (Chinese pharmacopoeia 2020 edition, four-part rule 2204).
The volatile oil content of the product is 0.18% -0.35% (ml/g).
The scutellarin and rosmarinic acid are measured by high performance liquid chromatography (general rule 0512 in the year of Chinese pharmacopoeia 2020).
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (column length of 150mm, inner diameter of 2.1mm, particle diameter of 1.6 μm) as chromatographic column; acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, and performing gradient elution according to Table 3; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 330nm, and the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peak.
TABLE 3 elution gradient for high performance liquid chromatography
Preparing control solution, precisely weighing appropriate amounts of scutellarin control and rosmarinic acid control, adding 70% methanol to obtain mixed solution containing 50 μg of scutellarin and 80 μg of rosmarinic acid per 1ml, and shaking.
Preparing test solution, namely taking 0.1g of each of batch 1 (comprising batch 1-1 and batch 1-2), batch 2 (comprising batch 2-1 and batch 2-2) and batch 3 (comprising batch 3-1 and batch 3-2) of the perilla leaf intermediate, precisely weighing, placing the materials into a conical flask with a plug, precisely adding 20ml of 70% methanol, weighing, performing ultrasonic treatment (with power of 250W and frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the loss weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
The measurement method comprises precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
The product contains scutellarin (C) 21 H 18 O 12 ) Between 0.18% and 1.00%, and contains rosmarinic acid (C) 18 H 16 O 8 ) The content of the active components is 0.18 to 2.00 percent.
1.3.2 results of the study
The results of the content of scutellarin, rosmarinic acid and volatile oil are shown in Table 4. The characteristic patterns of the intermediates of the perilla leaf and the patterns of the reference substances (scutellarin and rosmarinic acid) of different batches are shown in fig. 7, wherein 1-1 (2) in fig. 7 corresponds to batch 1-1,1-2 (2) corresponds to batch 1-2,2-1 (2) corresponds to batch 2-1,2-2 (2) corresponds to batch 2-2,3-1 (2) corresponds to batch 3-1,3-2 (2) corresponds to batch 3-2, and T (2) corresponds to scutellarin reference substance and rosmarinic acid reference substance. As can be seen from table 4 and fig. 7, the contents of scutellarin, rosmarinic acid and volatile oil in the batch 1, batch 2 and batch 3 of the perilla leaf intermediates all meet the standard specification without obvious change, and the stability is good.
TABLE 4 content of scutellarin, rosmarinic acid and volatile oils in different batches of Perilla leaf intermediate
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2. Stability between different batches of Perilla leaf formula particles
2.1 thin layer authentication
2.1.1 methods of investigation
(1) Respectively taking 0.5g of each of batch 1, batch 2 and batch 3 of the perilla leaf formula particles, respectively adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 2ml of methanol into residues to dissolve the residues to obtain a sample solution. And adding 1g of perilla leaf reference medicinal material, adding 50ml of water, boiling for 30 minutes, filtering, evaporating filtrate to dryness, adding 25ml of methanol into residues, and preparing a reference medicinal material solution by the same method. Test by thin layer chromatography (rule 0502 of four parts of chinese pharmacopoeia 2020 edition), T:16 ℃, RH:50%, sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-formic acid-water (9:0.5:1:0.5) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, and inspecting under ultraviolet lamp (365 nm).
(2) The volatile oil of the perilla leaf formula particles in batch 1, batch 2 and batch 3 is respectively taken, and n-hexane is respectively added to prepare solutions with 10 mu l of each 1ml of the volatile oil as a test solution. And adding n-hexane into the perillaldehyde reference substance to prepare a solution with the concentration of 0 mu l per lml as the reference substance solution. Test by thin layer chromatography (rule 0502 of four parts of chinese pharmacopoeia 2020 edition), T:20 ℃, RH:60%, sucking 5-10 μl of the sample solution and 2 μl of the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with n-hexane-ethyl acetate (15:1) as developing agent, taking out, air drying, and spraying dinitrophenylhydrazine ethanol solution.
2.1.2 results of the study
The results of the thin-layer identification patterns of the perilla leaf formula particles and the perilla leaf control medicinal materials in different batches are shown as A in fig. 8, the thin-layer identification patterns of the volatile oil and the perillaldehyde control substances in the perilla leaf formula particles in different batches are shown as B in fig. 8, and 1 in A in fig. 8 corresponds to batches 1 and 2, and 3 corresponds to batches 2 and 3, and S corresponds to the perilla leaf control medicinal materials. 1 in fig. 8 corresponds to lot 1, 2 corresponds to lot 2, 3 corresponds to lot 3, and T corresponds to the perillaldehyde control.
As can be seen from a in fig. 8, in the sample chromatograms of batch 1, batch 2 and batch 3 of the perilla leaf recipe granules, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference medicinal material, and the stability is good without significant difference. As can be seen from fig. 8B, in the sample chromatograms of batch 1, batch 2 and batch 3 of the perilla leaf recipe granules, spots with the same color appear at the positions corresponding to the chromatogram of the reference substance, no significant difference exists, and the stability is good. From this, the difference between different batches of the perilla leaf formulation granule prepared by the quality control method of the perilla leaf formulation granule in the above embodiment is small, i.e. the stability is good.
2.2 characteristic Spectrum
2.2.1 methods of research
[ characteristic map ] is measured by high performance liquid chromatography (four-part rule 0512 in the year of the Chinese pharmacopoeia 2020).
[ chromatographic conditions and System applicability test ] octadecylsilane chemically bonded silica was used as a filler (column length: 150mm, inner diameter: 2.1mm, particle size: 1.6 μm, waters CORTECS T3 column); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the specifications in Table 1; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength was 215nm. The number of theoretical plates should be not less than 3000 calculated as rosmarinic acid peak.
Preparation of reference solution 0.3g of folium Perillae reference material is put into a conical flask with a plug, 25ml of water is added, the mixture is heated and decocted for 30 minutes, cooled, shaken well and filtered, and the subsequent filtrate is taken as reference solution of reference material. And (3) taking a proper amount of scutellarin reference substance and rosmarinic acid reference substance, precisely weighing, and respectively adding methanol to prepare solutions containing 50 mug of scutellarin and 80 mug of rosmarinic acid per lml as reference substance solutions of the reference substances.
Preparation of sample solution preparation of batch 1 (including batch 1-1 and batch 1-2), batch 2 (including batch 2-1 and batch 2-2), batch 3 (including batch 3-1 and batch 3-2) of perilla leaf formula particles 0.1g each, precisely weighing, placing in a conical flask with a plug, precisely adding 70% methanol 20ml, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the loss weight with 70% methanol, shaking, filtering, and collecting the subsequent filtrate.
The measurement method comprises precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement to obtain the final product shown in Table 5.
4 characteristic peaks should be present in the chromatogram and correspond to the retention time of 4 characteristic peaks in the chromatogram of the reference substance of the control medicinal material; wherein peak 3 is scutellarin, peak 4 (S) is rosmarinic acid, and peak 3 and peak 4 should correspond to retention time of scutellarin and rosmarinic acid reference peak. The peak corresponding to the rosmarinic acid reference is S peak, and the relative retention time of the characteristic peaks 1,2 and S peak is calculated, wherein the relative retention time is within + -10% of the specified value. The specified value is: 0.29 (Peak 1), 0.45 (Peak 2).
2.2.2 results of the study
The characteristic spectrum of the perilla leaf formula granule, the spectrum results of the perilla leaf control medicinal materials and the reference substances (scutellarin and rosmarinic acid) of different batches are shown in fig. 9, 1-1 (4) in fig. 9 corresponds to batches 1-1,1-2 (4) corresponds to batches 1-2,2-1 (4) corresponds to batches 2-1,2-2 (4) corresponds to batches 2-2,3-1 (4) corresponds to batches 3-1,3-2 (4) corresponds to batches 3-2, T (2) corresponds to the scutellarin reference substance and the rosmarinic acid reference substance, and S (4) corresponds to the perilla leaf control medicinal materials.
The relative retention time of characteristic patterns of the perilla leaf formula granules in different batches is shown in table 5, and as can be seen from table 5 and fig. 9, characteristic patterns of the perilla leaf formula granules in batch 1, batch 2 and batch 3 all show 4 characteristic peaks, and the retention time corresponds to the retention time of 4 characteristic peaks in the control medicinal material reference object chromatograph respectively. Wherein peak 3 and peak 4 correspond to the retention time of scutellarin and rosmarinic acid reference peak respectively. The relative retention time of peak 1 and peak 2 is within + -10% of the prescribed value. In conclusion, the perilla leaf formula granules in different batches have small difference, namely good stability.
The detailed data are as follows:
TABLE 5 relative retention times of different batches of perilla leaf formulation particles
| Batch of | Peak 1 | Peak 2 | Peak 3 | Peak 4 (S) |
| Specified value | 0.29±10％ | 0.45±10％ | Scutellarin (scutellarin) | Rosmarinic acid |
| 1-1 | 0.296 | 0.447 | / | 1.000 |
| 1-2 | 0.296 | 0.447 | / | 1.000 |
| 2-1 | 0.307 | 0.483 | / | 1.000 |
| 2-2 | 0.307 | 0.483 | / | 1.000 |
| 3-1 | 0.306 | 0.482 | / | 1.000 |
| 3-2 | 0.306 | 0.482 | / | 1.000 |
| RSD(％) | 1.80 | 3.90 | / | / |
2.3 content determination
2.3.1. Research method
[ MEANS FOR SOLVING ] volatile oil is measured by volatile oil assay (Chinese pharmacopoeia 2020 edition, four-part rule 2204).
The volatile oil content of the product is 0.18% -0.35% (ml/g).
The scutellarin and rosmarinic acid are measured by high performance liquid chromatography (four general rules 0512 in the year of Chinese pharmacopoeia 2020).
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (column length of 150mm, inner diameter of 2.1mm, particle diameter of 1.6 μm) as chromatographic column; acetonitrile as mobile phase a and 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution was performed as specified in table 5; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 330nm, and the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peak.
Preparing control solution, precisely weighing appropriate amounts of scutellarin control and rosmarinic acid control, adding 70% methanol to obtain mixed solution containing 50 μg of scutellarin and 80 μg of rosmarinic acid per 1ml, and shaking.
Preparation of sample solution taking 0.1g each of batch 1 (comprising batch 1-1 and batch 1-2), batch 2 (comprising batch 2-1 and batch 2-2), batch 3 (comprising batch 3-1 and batch 3-2) of perilla leaf formula particles, precisely weighing, placing into a conical flask with a plug, precisely adding 70% methanol 20ml, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the loss weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
The measurement method comprises precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
The product contains scutellarin (C) per 1g 21 H 18 O 12 ) 1.5 mg-10.0 mg of rosmarinic acid (C) 18 H 16 O 8 ) Should be 1.5mg to 20.0mg.
2.3.2 results of the study
The results of the content of scutellarin, rosmarinic acid and volatile oil are shown in Table 6. The characteristic spectrum of the perilla leaf formula granules and the spectrum of the reference substances (scutellarin and rosmarinic acid) of different batches are shown in figure 10, wherein 1-1 (2) in figure 10 corresponds to batch 1-1,1-2 (2) corresponds to batch 1-2,2-1 (2) corresponds to batch 2-1,2-2 (2) corresponds to batch 2-2,3-1 (2) corresponds to batch 3-1,3-2 (2) corresponds to batch 3-2, and T (2) corresponds to scutellarin reference substance and rosmarinic acid reference substance. From this, the content difference of scutellarin, rosmarinic acid and volatile oil among the batch 1, batch 2 and batch 3 of the perilla leaf formula particles is small, i.e. the stability is high.
TABLE 6 content of scutellarin, rosmarinic acid and volatile oil in different batches of Perilla leaf formula granules
(2) Uniformity test between different samples of the same batch
1. Homogeneity test between different samples within the same batch of Perilla leaf intermediate
The research method comprises the following steps: and respectively carrying out uniformity tests on different samples in the same batch of the purple perilla leaf intermediate, namely batch 1, batch 2 and batch 3, wherein the test method of thin layer identification of the purple perilla leaf intermediate is 1.1, the test method of characteristic spectrum is 1.2, the test method of content measurement of scutellarin, rosmarinic acid and volatile oil is 1.3, the properties are described according to the actual properties of the samples and the samples of historical batches, the extract content is measured according to the alcohol-soluble extract measurement method of 2201 in the fourth rule of the 2020 edition of Chinese pharmacopoeia, and the moisture inspection is measured according to the moisture measurement method of 0832 in the fourth rule of the 2020 edition of Chinese pharmacopoeia. Test items and the results are shown in tables 7-9.
TABLE 7 uniformity between different samples of batch 1 of perilla leaf intermediate
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TABLE 8 uniformity between different samples of batch 2 of perilla leaf intermediate
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TABLE 9 uniformity between different samples of batch 3 of perilla leaf intermediate
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Study results: as is clear from the experimental results in tables 7 to 9, the uniformity of the leaf intermediate of Perilla frutescens in the same production lot was high.
2. Uniformity between different samples within the same batch of perilla leaf formulation particles
The research method comprises the following steps: and respectively carrying out uniformity tests on different samples in the same batch of the perilla leaf formula particles in batches 1, 2 and 3, wherein the test method for identifying the thin layer of the perilla leaf formula particles is 2.1, the test method for measuring the contents of scutellarin, rosmarinic acid and volatile oil is 2.2, the test method for measuring the contents of scutellarin, rosmarinic acid and volatile oil is 2.3, the description of the characteristics is described in 2020 edition according to the actual characteristics of the samples and the samples of historical batches, the extract content is measured according to the method for measuring 2201 alcohol-soluble extract in the fourth general rule of the Chinese pharmacopoeia 2020, the granularity inspection is measured according to the method for measuring the 0982 granularity of the fourth general rule of the Chinese pharmacopoeia 2020, and the moisture inspection is measured according to the 0832 moisture measurement method of the fourth general rule of the Chinese pharmacopoeia 2020. Test items and the results are shown in tables 10-12.
TABLE 10 uniformity between different samples of batch 1 of perilla leaf formulation particles
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TABLE 11 uniformity between different samples of batch 2 of perilla leaf formulation particles
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TABLE 12 uniformity among different samples of batch 3 of perilla leaf formulation particles
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Study results: as can be seen from the experimental results of tables 10 to 12, the uniformity of the perilla leaf formulation granules in the same production lot is high.
In conclusion, the application can improve the accuracy of true and false identification and quality evaluation by simultaneously measuring a plurality of characteristic indexes of the perilla leaf decoction pieces, the perilla She Zhongjian body and the perilla leaf formula particles, thereby not only ensuring the authenticity of the perilla leaf decoction pieces, the perilla She Zhongjian body and the perilla leaf formula particles, but also ensuring small difference (namely high stability) of the perilla She Zhongjian body and the perilla leaf formula particles among different production batches; the perilla She Zhongjian body and the perilla leaf formula granules of the same production batch have good uniformity. Thereby further optimizing the preparation process of the perilla leaf decoction pieces, the perilla She Zhongjian body and the perilla leaf formula granules. The whole process quality detection method has strong specificity and good repeatability, and can effectively control the quality of each link in the preparation process of the perilla leaf formula particles. In addition, the qualification rate of the perilla She Zhongjian body and the perilla leaf formula particles prepared by the quality control without adopting the whole process quality detection method is uncontrollable, but the qualification rate of the perilla She Zhongjian body and the perilla leaf formula particles among different production batches prepared by adopting the whole process quality control method and among different samples of the same production batch can be always controlled to be 100 percent.
It should be noted that the embodiments of the present invention are preferred and not limited in any way, and any person skilled in the art may make use of the above-disclosed technical content to change or modify the same into equivalent effective embodiments without departing from the technical scope of the present invention, and any modification or equivalent change and modification of the above-described embodiments according to the technical substance of the present invention still falls within the scope of the technical scope of the present invention.
Claims (6)
1. The whole process quality detection method in the preparation of the perilla leaf formula particles is characterized by comprising a quality detection method of S1 perilla leaf decoction pieces, a quality detection method of S2 perilla leaf intermediates and a quality detection method of S3 perilla leaf formula particles;
in the quality detection of S1, S2 and S3, scutellarin and rosmarinic acid are used as characteristic indexes, and volatile oil, scutellarin and rosmarinic acid are used as content indexes for detection;
s1, a quality detection method of perilla leaf decoction pieces comprises the following steps:
and (3) detecting characteristic indexes: the method for determining the scutellarin solution, the rosmarinic acid solution and the elemene solution serving as reference substance solutions, the solution obtained by decocting the perilla leaf reference medicinal material serving as reference substance solution, the solution of perilla leaf decoction piece powder serving as sample solution and the ultra-high performance liquid chromatography comprises the following steps: octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.6 mu m; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid solution is taken as a mobile phase B; gradient elution is carried out for 0-4 min,17% of A and 83% of B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm; the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peak; precisely sucking 1 μl of reference substance solution of reference substance, 1 μl of reference substance solution of folium Perillae reference medicinal material and 1 μl of sample solution respectively, and measuring with liquid chromatograph; analyzing and comparing the characteristic patterns of the reference substance solution and the sample solution, wherein the characteristic patterns of the sample solution should show 5 characteristic peaks and correspond to the retention time of 5 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the scutellarin reference peak, the characteristic peak 4 should correspond to the retention time of the rosmarin reference peak, the characteristic peak 5 should correspond to the retention time of the elemene reference peak, the characteristic peak corresponding to the rosmarin reference is the S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within + -10% of the specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.44;
And (3) content index detection: the volatile oil content of the perilla leaf decoction pieces is not less than 0.40% ml/g measured by volatile oil measurement method; the method comprises the steps of taking a scutellarin solution and a rosmarinic acid solution as reference substance solutions, taking a solution of perilla leaf decoction piece powder as a test substance solution, and determining that the mass percent of scutellarin in the perilla leaf decoction pieces is not less than 0.08% and the mass percent of rosmarinic acid is not less than 0.36% by high performance liquid chromatography;
s2, a quality detection method of a perilla leaf intermediate comprises the following steps:
and (3) detecting characteristic indexes: the method for determining the scutellarin solution and rosmarinic acid solution by using the scutellarin solution and rosmarinic acid solution as reference substance solutions, the solution obtained by decocting the perilla leaf reference medicinal material as reference substance solution, the solution of the intermediate of the perilla leaf as sample solution and the ultra-high performance liquid chromatography comprises the following steps: octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.6 mu m; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0 to 4min, the concentration of the phosphoric acid solution is 17 percent A, and the concentration of the phosphoric acid solution is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength is 215nm, and the number of theoretical plates is not less than 3000 calculated according to rosmarinic acid peaks; precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement; analyzing and comparing the characteristic patterns of the reference substance solution and the sample solution, wherein the characteristic patterns of the sample solution should show 4 characteristic peaks and correspond to the retention time of the 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; wherein the characteristic peak 3 should correspond to the retention time of the characteristic peak of the scutellarin reference substance, the characteristic peak 4 should correspond to the retention time of the reference substance peak of the rosmarinic acid reference substance, the characteristic peak corresponding to the rosmarinic acid reference substance is taken as an S peak, the relative retention time of the characteristic peak 1, the characteristic peak 2 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, the specified value of the relative retention time of the characteristic peak 1 is 0.29, and the specified value of the relative retention time of the characteristic peak 2 is 0.45;
And (3) content index detection: the volatile oil content of the purple perilla leaf intermediate measured by a volatile oil measuring method is 0.18% -0.35% ml/g; the method comprises the steps of taking a scutellarin solution and a rosmarinic acid solution as reference substance solutions, taking a solution of perilla leaf decoction piece powder as a sample solution, and measuring the mass percent of scutellarin in a perilla leaf intermediate by a high performance liquid chromatography to be 0.18-1.00%, wherein the mass percent of rosmarinic acid is 0.18-2.00%;
s3, a quality detection method of perilla leaf formula particles comprises the following steps:
the feature index detection is the same as S2;
and (3) content index detection: the volatile oil content of the purple perilla leaf intermediate measured by a volatile oil measuring method is 0.18% -0.35% ml/g; the scutellarin solution and the rosmarinic acid solution are used as reference substance solutions, the solution of the perilla leaf decoction piece powder is used as a sample solution, and the content of the scutellarin in each 1g of the perilla leaf formula particles is 1.5-10.0 mg and the content of the rosmarinic acid in each 1g of the perilla leaf formula particles is 1.5-20.0 mg measured by a high performance liquid chromatography;
in step S1 and step S2, a sample solution for feature index detection is prepared as follows: about 0.3g of perilla leaf decoction piece powder is taken, precisely weighed, placed in a conical bottle with a plug, precisely added with 25ml of 70% methanol, sealed, weighed, subjected to ultrasonic treatment for 30 minutes at the power of 250-300W and the frequency of 40kHz, cooled, weighed again, complemented with 70% methanol by the weight of loss, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the perilla leaf decoction piece.
2. The method for detecting the quality of the whole process in preparing the perilla leaf formula granules according to claim 1, wherein the step of the method for detecting the quality of the S1 perilla leaf decoction pieces further comprises the following steps: s0 quality detection method of folium Perillae medicinal material.
3. The method for detecting the quality of the whole process in the preparation of the perilla leaf formula granules according to claim 1, wherein the preparation of the reference solution is as follows: and (3) taking appropriate amounts of scutellarin reference substance solution, rosmarinic acid reference substance solution, elemene reference substance solution and perilla leaf reference substance solution, precisely weighing, and adding 70% methanol to prepare solutions containing 50 mug, 80 mug and 90 mug in each 1 ml.
4. The method for detecting the quality of the whole process in the preparation of the perilla leaf formula granules according to claim 1, wherein in the content index detection of S1, S2 and S3:
the high performance liquid chromatography determination method comprises the following steps: octadecylsilane chemically bonded silica is used as a filling agent of a chromatographic column; acetonitrile solution is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, gradient elution is carried out, the time is 0-4 min, the time is 17 percent A, and the time is 83 percent B; 4-5 min, 17-15% of A and 83-85% of B; 5-6 min, 15-23% of A and 85-77% of B; 6-8 min,23% of A and 77% of B; 8-11 min, 23-60% of A, 77-40% of B; 11-12 min,60% of A and 40% of B; 12-13 min, 60-100% of A, 40-0% of B; 13-18 min,100% A; the detection wavelength is 330nm; column temperature 40 ℃; the flow rate is 0.3ml per minute, and the theoretical plate number is not less than 3000 calculated according to rosmarinic acid peak; and respectively precisely sucking 1 μl of the reference substance solution and 1 μl of the sample solution, and injecting into a high performance liquid chromatograph for determination to obtain the contents of scutellarin and rosmarinic acid.
5. The method for detecting the quality of the whole process in the preparation of the perilla leaf formula granules according to claim 4, wherein the preparation of the reference solution is as follows: taking scutellarin as reference substance and rosmarinic acid as reference substance, precisely weighing, adding 70% methanol to obtain mixed solution containing 50 μg and 80 μg respectively per 1ml, and shaking.
6. The method for detecting the quality of the whole process in the preparation of the perilla leaf formula granules according to claim 1, wherein the preparation of the reference solution is as follows: taking appropriate amounts of scutellarin reference substance solution and rosmarinic acid reference substance solution, precisely weighing, and respectively adding methanol to obtain 50 μg/1 ml scutellarin-containing solution and 80 μg/rosmarinic acid-containing solution; taking 0.3g of perilla leaf reference medicinal material powder, precisely weighing, placing into a conical flask with a plug, adding 25ml of water, heating and decocting for 30 minutes, cooling, shaking uniformly, filtering, and collecting the subsequent filtrate;
the preparation of the sample solution is as follows: taking 0.1g of perilla leaf intermediate, precisely weighing, placing into a conical flask with a plug, precisely adding 20ml of 70% methanol, weighing, performing ultrasonic treatment at the power of 250W and the frequency of 40kHz for 30 minutes, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the perilla leaf intermediate.
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