CN117695232A - Preparation process of medicinal prescription two-instrument decoction granules and quality standard detection method - Google Patents

Preparation process of medicinal prescription two-instrument decoction granules and quality standard detection method Download PDF

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CN117695232A
CN117695232A CN202311720162.8A CN202311720162A CN117695232A CN 117695232 A CN117695232 A CN 117695232A CN 202311720162 A CN202311720162 A CN 202311720162A CN 117695232 A CN117695232 A CN 117695232A
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instrument
decoction
solution
peak
water
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郑明善
于跃
郎明越
呼德日
李喜玲
闵俊哲
康东周
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Yanbian University
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Yanbian University
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Abstract

The invention discloses a preparation process of a granule decocted by two instruments towards a doctor, which comprises the following steps: extracting radix rehmanniae Preparata and Corni fructus with water, concentrating, and lyophilizing to obtain two-instrument decoction lyophilized powder; mixing the two-instrument decocted freeze-dried powder and auxiliary materials uniformly, adding a wetting agent, preparing primary product particles through a sieve, and drying to obtain two-instrument decocted particles; the invention also discloses a detection method of the two-instrument decoction granules for the medical prescription, which comprises the steps of property, appearance inspection, granularity, moisture, solubility, loading difference, extract, thin-layer chromatography identification, fingerprint identification and content measurement; the invention determines the granule forming process by screening out the optimal auxiliary materials and the mixture ratio thereof for preparing the two-instrument decoction granules, and simultaneously checks various indexes of the two-instrument decoction granules so as to realize the quality control of the preparation.

Description

Preparation process of medicinal prescription two-instrument decoction granules and quality standard detection method
Technical Field
The invention relates to the field of national prescription quality control, in particular to a preparation process of a medicinal prescription decoction granule and a quality standard detection method.
Background
The two decoction are recorded in the ancient books of the medical science of the new "Dong medical insurance" and "Chao Yao Zhi" and are the traditional Chao medical prescription. Prepared rehmannia root and dogwood, according to the following proportion of 2: 1. According to the theory of medicine, the prepared rehmannia root and the dogwood are combined to have the functions of tonifying kidney and replenishing essence, and are used for treating the cold pain syndrome of the jade stems, and at present, few reports are given for the decoction of the two instruments of the traditional medicine prescription.
The two materials of prepared rehmannia root and dogwood fruit of the two-instrument decoction formula are both received in Chinese pharmacopoeia (2020 edition). The prepared rehmannia root is a processed product of root of rehmannia Rehmanniae glutinosa Libosch belonging to the Scrophulariaceae, which is slightly warm in nature and sweet in taste. It enters liver and kidney meridians. Efficacy: nourishing yin and tonifying blood. It can be used for treating deficiency of yin, blood deficiency, debility of waist and knee, fatigue, bone steaming, spermatorrhea, metrorrhagia, menoxenia, diabetes, scanty urine, deafness, and dizziness. Mainly contains cyclic ether terpene glycosides. The fructus Corni is dried mature pulp of Cornus officinalis of Cornaceae, and has slightly warm and sour taste. It enters liver and kidney meridians. Efficacy: tonify liver and kidney, astringe and arrest spontaneous emission. Can be used for treating dizziness, tinnitus, soreness of waist and knees, sexual impotence, spermatorrhea, enuresis, frequent urination, metrorrhagia, leukorrhagia, excessive sweat, deficiency, internal heat, and diabetes. The chemical components mainly comprise reducing sugar, polysaccharide, organic acid, phenols, glycosides, iridoid glycoside, saponin and other organic substances. The prepared rehmannia root in the two decoction formulas can tonify true yin, and traditional Chinese medicine doctors take prepared rehmannia root as kidney-tonifying essential medicine, for example, the traditional Chinese medicine is suitable for Zhangjing Yuan for dispersing yin deficiency, the prepared rehmannia root is not sufficient for gathering, the prepared rehmannia root is not sufficient for generating fire due to yin deficiency, and the prepared rehmannia root is not sufficient for lowering the weight; for restlessness due to yin deficiency, the silence of non-prepared rehmannia root is insufficient to relieve the symptoms; shan Zhu Yu is sour and astringent and combined with shan Zhu Yu to nourish yin and replenish essence. Ancient doctors treated kidney-yin deficiency with the idea of tonifying and applying, for example, yetianshi thought that it was not general but not astringent, and that it was not astringent but not essential. The only non-fixation is free and empty, and the only fixation is the convergence is too excessive, so that the drug can guide the astringent drug to arrest spontaneous emission, so that the drug can better play the role of tonifying kidney. According to the theory of the traditional Chinese medicine, the prepared rehmannia root and the dogwood have the functions of tonifying kidney and replenishing essence, can be used for treating symptoms such as internal heat due to kidney yin deficiency, clinically and effectively delay the progress of chronic renal failure in early and middle stages, remarkably improve the renal function, regulate lipid metabolism disorder, remarkably alleviate or eliminate clinical symptoms, and are effective prescriptions for improving the life quality of patients.
Disclosure of Invention
The invention designs and develops a preparation process of a two-instrument decoction granule towards a medical prescription, and aims to determine a granule forming process by screening out optimal auxiliary materials and proportions of the optimal auxiliary materials for preparing the two-instrument decoction granule.
The invention designs and develops a method for detecting quality standards of two-instrument decoction granules towards a doctor, and aims to detect various indexes of the two-instrument decoction granules according to requirements of Chinese pharmacopoeia (2020 edition), and a high performance liquid chromatography is adopted to establish a simultaneous measurement technology of contents of 5-HMF, protocatechuic acid, morroniside, loganin, swertiside and acteoside in the two-instrument decoction granules so as to realize quality control of the preparation.
The technical scheme provided by the invention is as follows:
a preparation process of a medicinal prescription decoction granule comprises the following steps:
extracting radix rehmanniae Preparata and Corni fructus with water, concentrating, and lyophilizing to obtain two-instrument decoction lyophilized powder;
mixing the two-instrument decocted freeze-dried powder and auxiliary materials uniformly, adding a wetting agent, preparing primary product particles through a sieve, and drying to obtain the two-instrument decocted particles;
Wherein, the mass ratio of the prepared rehmannia root to the dogwood is 2:1, the mass ratio of the two-instrument decocted freeze-dried powder to the auxiliary materials is 1:2, the auxiliary materials comprise dextrin, starch and mannitol.
Preferably, the water extraction process comprises: adding 6-10 times of water into the medicinal materials, and decocting for 1-3 times, wherein each time of decoction lasts for 0.5-1.5 hours.
Preferably, the mass ratio of the two-meter fried freeze-dried powder to the dextrin to the starch to the mannitol is as follows: 1:0.5 to 1:0.25 to 1:0.25 to 0.75.
Preferably, the wetting agent is an ethanol solution with the concentration of 80% -95%.
Preferably, the drying temperature after the preparation of the primary product particles is 60-69 ℃, 70-79 ℃ or 80-89 ℃; and the drying time is 2-4 h.
Preferably, adding 8 times of water into prepared rehmannia root and dogwood, decocting for 2 times, each time for 1.5 hours, filtering, concentrating, and freeze-drying to obtain two-instrument decoction freeze-dried powder;
the two instruments are decocted to freeze-dried powder, dextrin, starch and mannitol according to the mass ratio of 1:1:0.5: and (3) after uniformly mixing 0.5, adding an ethanol solution with the concentration of 90%, granulating by a 10-mesh sieve, and drying at 70 ℃ to obtain the two-instrument decoction granules.
A method for detecting quality standard of granule decocted in two instruments for medical prescription comprises properties, appearance inspection, granularity, water content, solubility, difference of loading, extract, thin layer chromatography identification, fingerprint identification and content measurement;
wherein, the content measurement process comprises:
preparing a test solution: adding methanol into the two decoction particles to fix volume, performing ultrasound, supplementing the weight loss with methanol, shaking uniformly, filtering, and collecting the subsequent filtrate to obtain the sample solution;
preparing a mixed reference substance solution: taking a proper amount of 5-HMF, protocatechuic acid, morroniside, loganin, swertisin and acteoside as reference substances, adding methanol for dissolving, and then fixing the volume to obtain mixed reference substance solutions with the concentration of 52.5 mug/mL, 12.5 mug/mL, 115.0 mug/mL, 67.5 mug/mL, 47.5 mug/mL and 27.5 mug/mL respectively;
preparing a negative sample solution: respectively taking negative samples prepared by removing radix rehmanniae Preparata and Corni fructus, adding methanol to constant volume, performing ultrasound, supplementing reduced weight with methanol, shaking, filtering, and collecting subsequent filtrate to obtain the negative sample solution;
and respectively precisely sucking the sample solution, the mixed reference substance solution and the negative sample solution, injecting the sample solution into a high performance liquid chromatograph, and measuring by gradient elution to obtain the content.
Preferably, the chromatographic conditions are: using chromatographic columns as Promosil C 18 Is used as a stationary phase, the column temperature is 25 ℃, the detection wavelength is 260nm, the flow rate is 0.6mL/min, the analysis time is 80min, and 10 mu L of sample injection amount is adopted for injection into a high performance liquid chromatograph; and
the elution procedure for the gradient elution was as follows:
at 0min, mobile phase A was 95% formic acid in water and mobile phase B was 5% acetonitrile;
at 20 minutes, mobile phase A was 91% formic acid in water and mobile phase B was 9% acetonitrile;
at 35 minutes, mobile phase A was 85.5% formic acid in water and mobile phase B was 14.5% acetonitrile;
at 50 minutes, mobile phase A was 82.5% formic acid in water and mobile phase B was 17.5% acetonitrile;
at 62 minutes, mobile phase A was 77% formic acid in water and mobile phase B was 23% acetonitrile;
at 67 minutes, mobile phase A was 77% formic acid in water and mobile phase B was 23% acetonitrile;
at 77 minutes, mobile phase A was 71% formic acid in water and mobile phase B was 29% acetonitrile;
at 80 minutes, mobile phase A was 5% formic acid in water and mobile phase B was 95% acetonitrile;
the column temperature is 25 ℃; flow rate: 0.6mL/min; sample injection amount: 10 mu L.
Preferably, the fingerprint spectrum in the fingerprint spectrum identification has 19 shared fingerprint peaks, and the relative retention time is respectively:
peak No. 1: 0.368-0.370; peak No. 2: 0.512-0.514; peak No. 3: 0.553 to 0.556; peak No. 4: 0.609 to 0.613; peak No. 5: 0.657 to 0.662; peak No. 6: 0.710 to 0.712; peak No. 7: 0.759 to 0.760; peak No. 8: 0.830 to 0.832; peak No. 9: 0.854 to 0.857; peak No. 10: 0.875 to 0.879; peak No. 11: 0.920 to 0.923; peak No. 12 is a reference peak: 1.000; peak No. 13: 1.021 to 1.022; peak No. 14: 1.107 to 1.108; peak No. 15: 1.265 to 1.266; peak No. 16: 1.469 to 1.471; peak No. 17: 1.526 to 1.531; peak No. 18: 1.570-1.573; peak No. 19: 1.609 to 1.613.
Preferably, the thin layer chromatography identification process comprises:
adding 80% methanol into the two-instrument decoction particles, filtering, evaporating filtrate to dryness, dissolving residues in water, shaking and extracting with water saturated n-butanol, evaporating to dryness, dissolving residues in methanol, and taking the residues as a sample solution of the two-instrument decoction particles; preparing reference medicinal material solution from radix rehmanniae Preparata by the method for preparing sample solution; adding methanol as reference solution; taking a negative sample prepared by removing prepared rehmannia root, and preparing a negative control solution according to a preparation method of a sample solution; sucking 2 μl of each of the control solution and the control solution by thin layer chromatography, 5 μl of the test solution and 5 μl of the negative sample solution, and spotting on the same silica gel GF 254 Spreading ethyl acetate-methanol-formic acid-water at a ratio of 16:0.5:2.5:0.5 on the thin layer plate, taking out, air drying, spraying 10% sulfuric acid ethanol solutionAiring, heating to clear spots at 105 ℃, and inspecting under a 365nm ultraviolet lamp; in the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control sample, and no negative interference exists.
Taking two-instrument decoction particles, adding methanol, filtering, evaporating filtrate to dryness, and adding methanol into residues to dissolve the residues to obtain a sample solution of the two-instrument decoction particles; preparing a control medicinal material solution from a dogwood control medicinal material according to a preparation method of a sample solution; taking a morroniside reference substance and a loganin reference substance, and respectively adding methanol to prepare reference substance solutions; taking a negative sample prepared by removing dogwood, and preparing a negative control solution according to a preparation method of a sample solution; sucking 2 μl of each of the control solution and the control solution by thin layer chromatography, 5 μl of the test solution and 5 μl of the negative sample solution, and spotting on the same silica gel GF 254 Spreading chloroform-methanol as developing agent at a ratio of 2:1, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until spots are clear, and inspecting under 254nm ultraviolet lamp; in the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control sample, and no negative interference exists.
Compared with the prior art, the invention has the following beneficial effects:
1. the preparation process of the two-instrument decoction granules for the medical prescription provided by the invention adopts a water decoction method for extraction, and the optimal extraction process which is selected by optimization lays a foundation for the research of the dosage forms of the two-instrument decoction and provides scientific basis for industrial production;
2. based on the screened optimal extraction process, two-instrument fried freeze-dried powder is prepared, and early preparation is carried out for the forming process of the granules;
3. the quality standard of the two-instrument decoction granule is established, and the content measurement, the specificity experiment and the methodology investigation are carried out on the 5-HMF, the protocatechuic acid, the morroniside, the loganin and the acteoside in the two-instrument decoction granule by utilizing the HPLC content simultaneous measurement technology, so that the stability of the result is strong, the repeatability is good, and the accuracy is high; finally, according to the requirements of the general rule 0104 granule item in the edition 2020 of Chinese pharmacopoeia, the granularity, the moisture, the dissolubility, the loading difference and the extract of three batches of samples are checked, and the results all meet the regulations.
Drawings
Fig. 1 is a flow chart of a preparation process of the granule decocted by two instruments towards the medical prescription.
Fig. 2 shows the critical relative humidity of the granule decocted by two instruments towards the doctor according to the invention.
Fig. 3a is a chromatogram of a blank solution in a study of specificity of a granule decocted by two instruments towards a doctor according to the invention.
Fig. 3b is a chromatogram of a sample solution in a study of specificity of the granule decocted by two instruments towards the doctor.
Fig. 3c is a chromatogram of a mixed reference solution in a special investigation of the granule decocted by two instruments towards the medical prescription.
Fig. 3d is a chromatogram of a negative sample solution of prepared rehmannia root in a special investigation of granule decoction by two instruments towards the medical prescription.
Fig. 3e shows a chromatogram of a dogwood negative sample solution in a special investigation of two-instrument decoction granules towards the medical prescription.
FIG. 4a is a schematic diagram of a standard curve for 5-HMF assay according to the invention.
FIG. 4b is a schematic diagram of a standard curve for measuring the content of protocatechuic acid according to the invention.
Fig. 4c is a schematic diagram of a standard curve for determining the content of moenoside according to the present invention.
Fig. 4d is a schematic diagram of a standard curve for determining the content of loganin according to the invention.
Fig. 4e is a schematic diagram of a standard curve for determining the content of swertia glycoside according to the present invention.
FIG. 4f is a schematic diagram of a standard curve for determining the content of acteoside according to the present invention.
Fig. 5a shows a thin-layer identification chromatogram of radix rehmanniae Preparata in two-instrument decoction particles according to the present invention (1 is a calycoside reference substance, 2 is a radix rehmanniae Preparata reference substance, 3 is a 20230825-batch two-instrument decoction particle test sample, 4 is a 20230826-batch two-instrument decoction particle test sample, 5 is a 20230827-batch two-instrument decoction particle test sample, and 6 is a negative reference sample).
Fig. 5b shows a thin-layer identification chromatogram of dogwood in two-instrument decoction particles (1 is a morroniside reference substance, 2 is a loganin reference substance, 3 is a dogwood reference substance, 4 is a 20230825-batch two-instrument decoction particle test sample, 5 is a 20230826-batch two-instrument decoction particle test sample, 6 is a 20230827-batch two-instrument decoction particle test sample, and 6 is a negative reference sample) according to the present invention.
Fig. 6 is a fingerprint spectrum of a common mode according to the present invention.
FIG. 7 is a chromatogram of 10 batches of two-instrument decoction particles according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
As shown in fig. 1 to 4, the invention provides a preparation process of a decoction granule for two instruments towards a doctor, which comprises the following steps: extracting radix rehmanniae Preparata and Corni fructus with water, concentrating, and lyophilizing to obtain two-instrument decoction lyophilized powder; mixing the freeze-dried powder and auxiliary materials uniformly, adding a wetting agent, preparing primary product particles through a sieve, and drying to obtain the two-instrument decocted granules; wherein, the mass ratio of the prepared rehmannia root to the dogwood is 2:1, the mass ratio of the two-instrument decocted freeze-dried powder to the auxiliary materials is 1:2, auxiliary materials comprise dextrin, starch and mannitol.
The invention also provides a method for detecting the quality standard of the granule decocted by two instruments towards the medical prescription, which comprises the steps of detecting properties, appearance, granularity, moisture, dissolubility, difference in filling amount, extract and content measurement; wherein, the content measurement process comprises:
preparing a test solution: adding methanol into the two decoction particles to fix volume, performing ultrasound, supplementing the weight loss with methanol, shaking uniformly, filtering, and collecting the subsequent filtrate to obtain the sample solution; preparing a mixed reference substance solution: taking a proper amount of 5-HMF, protocatechuic acid, morroniside, loganin, swertisin and acteoside as reference substances, adding methanol for dissolving, and then fixing the volume to obtain mixed reference substance solutions with the concentration of 52.5 mug/mL, 12.5 mug/mL, 115.0 mug/mL, 67.5 mug/mL, 47.5 mug/mL and 27.5 mug/mL respectively;
preparing a negative sample solution: respectively taking negative samples prepared by removing radix rehmanniae Preparata and Corni fructus, adding methanol to constant volume, performing ultrasound, supplementing reduced weight with methanol, shaking, filtering, and collecting subsequent filtrate to obtain the negative sample solution;
and respectively precisely sucking the sample solution, the mixed reference substance solution and the negative sample solution, injecting the sample solution into a high performance liquid chromatograph, and measuring by gradient elution to obtain the content.
Examples
1. Instrument and materials
1. The experimental apparatus is shown in table 1.
Table 1 laboratory apparatus
2. Reagent and reagent
The medicines and reagents used in the experimental process are shown in tables 2 and 3, and the medicinal material decoction pieces used in the experimental process are purchased by each hospital drug bureau in Yanbian; the processed product of dried root tuber of rehmannia Rehmannia glutinosa Libosch, a figwort family plant, was identified by the professor Zheng Mingshan of Yanbian university. The Corni fructus is dried mature pulp of Corni fructus Cornus officinalis Sieb.et Zucc. The quality of the decoction pieces is detected according to the method specified under the item of the Chinese pharmacopoeia of 2020 edition, and the quality detection meets the requirements.
Table 2 experimental reagents
2. Preparation process
1. Research of extraction process
(1) Investigation of water absorption: as shown in Table 4, 80g of prepared rehmannia root, 40g of cornus officinalis, 120g of total weight, putting the prepared rehmannia root, 10 times of water in a container, soaking, confirming the soaking condition of decoction pieces every other hour, recording the water quantity which is not absorbed until the decoction pieces are completely soaked, and keeping the water quantity which is not absorbed until the water quantity which is not absorbed is not reduced, wherein the water absorption multiple can reach 1.50 after soaking for 4 hours.
TABLE 4 investigation of the water absorption of decoction pieces
(2) Inspection of the amount of water added: as shown in table 5, the water absorption samples are taken, all water is poured out, then water is added to observe that the water is not over the decoction pieces, the water adding amount of the over decoction pieces is 1.6 times, the condition that the heated water is lost in the decoction process and the water interface is 3-5 cm higher than the decoction piece interface is considered, at this time, when the water is 5cm higher than the water surface, the water adding multiple is 4.17 times, the water absorbing multiple is 1.50 times and is 5.67 times, namely the integral multiple is taken to be 6 times.
TABLE 5 investigation of the water addition of decoction pieces
(3) Orthogonal experiment design
(1) Selection of factor level: the different factor level design table is shown in Table 6, wherein the water addition multiple affecting the extraction effect of the two apparatuses is A (6 times, 8 times, 10 times), the decoction times are B (1 time, 2 times, 3 times), the decoction time is C (0.5 h, 1.0h, 1.5 h), and L is adopted 9 (3 4 ) The best extraction technique of two-instrument decoction is optimized in the orthogonal experiment.
TABLE 6 test factor level table
(2) Measurement of index component content: chromatographic conditions: by using Promosil C 18 (4.6 mm. Times.250 mm,5 μm); acetonitrile (B) -0.15% formic acid aqueous solution (A) is taken as a mobile phase, and gradient elution is carried out (0-20 min, 5-9% B, 20-35 min, 9-14.5% A, 35-50 min,14.5% B-17.5% B, 50-62 min,17.5% B-23% B, 62-67 min,23% B, 67-77 min,23% B-29% B, 77-80 min,29% B-95% B); the flow rate is 0.6mL/min; the detection wavelength is 260nm; column temperature: 25 ℃; sample injection amount: 10 mu L.
Preparation of a control solution: precisely weighing appropriate amount of acteoside, morroniside and loganin, adding methanol to obtain mixed reference solution containing acteoside 0.11mg, morroniside 0.46mg and loganin 0.27mg per 1mL, and shaking.
Preparation of test solution: precisely weighing 40g of prepared rehmannia root and 20g of dogwood, adding 8 times of water, soaking for 30min, decocting for 2 times, each time for 1.5h, filtering with a 200-mesh screen while the materials are hot, combining the two filtrates, concentrating to 150mL, and freeze-drying to obtain the two-instrument decocted freeze-dried powder. Precisely weighing about 1.0g of the two-meter decocted freeze-dried powder, adding methanol to a 25mL volumetric flask for constant volume, sealing, performing ultrasonic treatment for 30 minutes (frequency 50KHz and power 250W), standing, cooling, filtering by a 0.22 mu m microporous filter membrane, and taking a subsequent filtrate to obtain a two-meter decocted sample solution.
(4) Orthogonal experimental results: 40g of prepared rehmannia root and 20g of dogwood are weighed, and are tested according to an orthogonal test design table, and the table 7 is L 9 (3 4 ) Orthogonal experimental design results, table 8 shows analysis of variance results.
TABLE 8 analysis of variance
Note that: f (F) 0.05 (2，2)＝19.000
(5) Analysis of orthogonal results: from visual analysis results, the influence of different factors on the extraction process is C in turn>B>A，C 3 ＞C 1 ＞C 2 ，B 2 ＞B 3 ＞B 1 ，A 2 ＞A 1 ＞A 3 Judging by the comprehensive score mean value, and combining with analysis of variance to obtain the optimal process collocation C 3 B 2 A 2 . The analysis of variance results shows that in the water extraction process, the water adding times (A), the decoction times (B) and the decoction time (C) have no obvious influence on the extraction effect. Therefore, based on the principle of energy-saving green production, the influences of 3 factors on the paste yield and the content of each component of the two-instrument decoction are comprehensively analyzed, and the optimal extraction process is selected as C by combining visual and variance analysis results 3 B 2 A 2 I.e. 8 times of water, 2 times of decoction for 1.5h each time.
(6) And (3) optimal process verification: 40g of prepared rehmannia root and 20g of dogwood are respectively weighed and 3 parts of dogwood are added with 8 times of water for 2 times of decoction according to a determined extraction process, and each time is 1.5 hours. And measuring the content and the paste yield of the acteoside, morroniside and loganin in the extracting solution. As shown in Table 9, the results of the verification test show that under the extraction condition, the content and the ointment yield of the acteoside, the morroniside and the loganin are more stable, and the extraction process of the process is reasonable and feasible.
Table 9 process verification
2. Study of the Forming Process
40g of prepared rehmannia root and 20g of dogwood are taken, 8 times of water is added according to a determined extraction process, and the mixture is decocted for 2 times, and each time is 1.5 hours. Concentrating the two-instrument decoction extract properly, and freeze-drying to obtain the two-instrument decoction freeze-dried powder. The freeze-dried powder is used as a medicine for subsequent auxiliary material screening and granulation process research.
(1) Kinds of auxiliary materials
The auxiliary materials are selected according to the following steps: the proper auxiliary materials are selected according to the formability, bulk density, repose angle, dissolubility and hygroscopicity of the granules prepared by uniformly mixing the medicinal powder with the auxiliary materials.
The experimental method comprises the following steps: taking 4 parts of two decoction materials, and respectively mixing 2g of each decoction material with dextrin, lactose, starch and microcrystalline cellulose according to the ratio of 1:2 of the medicinal powder to the auxiliary materials, and measuring the formability, bulk density, repose angle, dissolution rate and hygroscopicity. And comprehensively evaluating the formability, bulk density, repose angle, dissolution rate and hygroscopicity as indexes, and optimizing the auxiliary materials.
Comprehensive index = (15/maximum formability value) ×formability value + (15/maximum bulk density value) ×bulk density value + (minimum angle of repose value×15)/angle of repose value + (20/maximum solubility value) ×solubility value + (minimum moisture absorption value×35)/moisture absorption value.
(1) Measurement of formability:
the prepared granules were sequentially passed through a first sieve and a fifth sieve, and the molding rate was regarded as an inspection index [ molding rate (%) =acceptable granule weight/total granule weight×100% ] (acceptable granules are granules which pass through the first sieve but cannot pass through the fifth sieve). The formability score calculation applies the formula: molding ratio score = (15/maximum moldability value) ×moldability value, and the result is shown in table 10.
Table 10 investigation of the Forming Rate of different types of auxiliary Material
(2) Determination of bulk Density
Bulk density, also known as apparent density or bulk density, refers to the mass of particles per unit volume. Bulk density the volume used refers to the total volume of the particles and their own voids and voids between particles. The large bulk density of the particles, i.e. small bulk volume, can indicate the degree of confidentiality of the particles and determine the volume fraction.
Placing qualified particles into a dry measuring cylinder, slightly vibrating, and reading the ml number (V) of the particles near the scale; and taking the qualified particles as M (g), wherein the ratio of the two is the bulk density. Bulk density=m/V ]. Bulk density score calculation applying the formula: bulk density score = (15/maximum bulk density value) ×bulk density value, and the result is shown in table 11.
Table 11 bulk Density investigation of different kinds of auxiliary Material
(3) Measurement of the angle of repose
Flowability is one of important properties of the granule, and the quality of flowability is related to the quality of the granule and the accuracy of dose division, and is commonly expressed by flow rate and repose angle in pharmacy. The smaller the angle of repose, the better the flow. Generally, the smaller the particle size or the wider the particle size distribution, the larger the angle of repose; and the particles with large and uniform particle size are easy to flow, and the repose angle is small.
3 funnels were connected in series and fixed at a height of 1cm on a horizontally placed piece of co-ordinate paper using a fixed funnel method, the particles were carefully poured into the uppermost funnel along the funnel wall until the tip of the cone of particles formed on the co-ordinate paper contacted the funnel opening, the diameter (2R) of the bottom of the cone was measured from the co-ordinate paper, and the [ angle of repose tanα=h/R ] was calculated, and the average was calculated 3 times. The repose angle score calculation applies the formula: the angle of repose score = (minimum angle of repose value x 15)/angle of repose value, the results are shown in table 12.
Table 12 investigation of angles of repose for different types of excipients
(4) Determination of dissolution Rate
1.0g of precisely weighed sample particles was added to a 10mL centrifuge tube (minimum scale: 0.1 mL) dried to a constant weight, 5mL of boiling water was precisely added, stirred and shaken for 5min, and centrifuged at 3000r/min for 15min, the supernatant was discarded, the residue was dried to a constant weight at 80℃and precisely weighed, and the melting rate [ melting rate (%) = melting particle weight/total particle weight×100% ], the melting score= (20/maximum melting value) ×melting value were calculated, and the measurement results were shown in Table 13.
TABLE 13 investigation of dissolution rates of different types of auxiliary Material
(5) Determination of hygroscopicity
The glass dryer, with supersaturated solution of sodium chloride at the bottom, was left at room temperature for 48 hours to equilibrate, at which point the relative humidity in the dryer was 75%. The sample of 0.5g was placed at the bottom of a 5mL flat weighing flask dried to a constant weight, and the flask was gently shaken to uniformly distribute the sample, precisely weighed, placed in a desiccator containing a supersaturated sodium chloride solution (the weighing flask lid was opened), weighed after 48 hours, and the moisture absorption rate [ moisture absorption rate (%) = (weight of powder after moisture absorption-weight of powder before moisture absorption)/weight of powder before moisture absorption×100% ] was calculated, and the moisture absorption rate score= (minimum moisture absorption rate value×35)/moisture absorption rate value was measured, and the measurement results were shown in table 14.
Table 14 investigation of hygroscopicity of different kinds of auxiliary materials
(6) Comprehensive scoring
As shown in table 15, the test results of the above items (1) to (5) were comprehensively evaluated.
Table 15 comprehensive score
The dextrin and the starch can be found to have higher scores according to the comprehensive scores of the data in the table, and the starch is suitable for being used as auxiliary materials; lactose is used as an auxiliary material, so that the viscosity is high, the hand is easy to adhere, and the molding is not performed; microcrystalline cellulose forms better but has more sediment when dissolved. Because the prepared rehmannia root has high viscosity in the two-instrument decoction, dextrin and starch are preferably selected as auxiliary materials. Because the original prescription has slight smell, sour, astringent and slightly bitter taste, mannitol is added as a corrective.
(2) Auxiliary material proportion
Taking 4 parts of two decoction materials, and uniformly mixing 2g of each part according to the proportion of 1:0.5:1:0.25, 1:0.5:1:0.5, 1:1:0.25:0.75, 1:1:0.5:0.5 and 1:1:1:0.5 of the medicinal powder to the dextrin, the starch and the mannitol. Preparing soft material with 90% ethanol, granulating with 10 mesh sieve, grading with 10 mesh sieve, drying at 70deg.C to constant weight, and measuring formability, bulk density, angle of repose, dissolution rate and hygroscopicity.
(1) Measurement of formability
The prepared granules were sequentially passed through a first sieve and a fifth sieve, and the molding rate was regarded as an inspection index [ molding rate (%) =acceptable granule weight/total granule weight×100% ] (acceptable granules are granules which pass through the first sieve but cannot pass through the fifth sieve). The formability score calculation applies the formula: molding ratio score = (15/maximum moldability value) ×moldability value, and the result is shown in table 16.
TABLE 16 Forming Rate score for different adjuvant ratios
(2) Determination of bulk Density
Placing qualified particles into a dry measuring cylinder, slightly vibrating, and reading the ml number (V) of the particles near the scale; and taking the qualified particles as M (g), wherein the ratio of the two is the bulk density. Bulk density=m/V ]. Bulk density score calculation applying the formula: bulk density score = (15/maximum bulk density value) ×bulk density value, and the result is shown in table 17.
TABLE 17 bulk Density score for different adjuvant ratios
(3) Measurement of the angle of repose
3 funnels were connected in series and fixed at a height of 1cm on a horizontally placed piece of co-ordinate paper using a fixed funnel method, the particles were carefully poured into the uppermost funnel along the funnel wall until the tip of the cone of particles formed on the co-ordinate paper contacted the funnel opening, the diameter (2R) of the bottom of the cone was measured from the co-ordinate paper, and the [ angle of repose tanα=h/R ] was calculated, and the average was calculated 3 times. The repose angle score calculation applies the formula: the angle of repose score = (minimum angle of repose value x 15)/angle of repose value, the results are shown in table 18.
TABLE 18 repose angle scores for different adjuvant ratios
(4) Determination of dissolution Rate
1.0g of precisely weighed sample particles was added to a 10mL centrifuge tube (minimum scale: 0.1 mL) dried to a constant weight, 5mL of boiling water was precisely added, stirred and shaken for 5min, and centrifuged at 3000r/min for 15min, the supernatant was discarded, the residue was dried to a constant weight at 80℃and precisely weighed, and the dissolution rate [ melting rate (%) = melting particle weight/total particle weight×100% ], melting score= (20/maximum melting value) ×melting value were calculated, and the measurement results are shown in Table 19.
Table 19 investigation of dissolution rates of different types of auxiliary materials
(5) Determination of hygroscopicity
The glass dryer, with supersaturated solution of sodium chloride at the bottom, was left at room temperature for 48 hours to equilibrate, at which point the relative humidity in the dryer was 75%. The sample of 0.5g was placed at the bottom of a 5mL flat weighing flask dried to a constant weight, and the flask was gently shaken to uniformly distribute the sample, precisely weighed, placed in a desiccator containing a supersaturated sodium chloride solution (the weighing flask lid was opened), weighed after 48 hours, and the moisture absorption rate [ moisture absorption rate (%) = (weight of powder after moisture absorption-weight of powder before moisture absorption)/weight of powder before moisture absorption×100% ] was calculated, and the moisture absorption rate score= (minimum moisture absorption rate value×35)/moisture absorption rate value was measured, and the measurement results were shown in table 20.
Table 20 hygroscopicity scores for different adjuvant ratios
(6) Comprehensive scoring
As shown in table 21, the test results of the above items (1) to (5) were comprehensively evaluated.
Table 21 comprehensive score
From the data composite scores in the table, it was found that when the powder was combined with dextrin, starch and mannitol at 1:1:0.5: the components are uniformly mixed and granulated according to the proportion of 0.5, the comprehensive score is higher, and the granule is suitable for being used as the optimal medicine auxiliary ratio of the two-instrument decoction granules.
(3) Wetting agent
Taking 4 parts of substance based on the weight of the substance, adding auxiliary materials of dextrin, starch and mannitol into each 2g of substance according to the ratio of 1:1:0.5:0.5, and uniformly mixing. Spraying ethanol with different concentrations, stirring and mixing for 10min, granulating, drying at 70deg.C, taking molding rate as investigation index, collecting granules capable of passing through No. 1 sieve and granules incapable of passing through No. five sieve as qualified products, and calculating the qualification rate. The measurement results are shown in Table 22.
Table 22 selection of wetting agents
The results show that the qualification rate is maximum when the wetting agent is 90% ethanol, so that 90% ethanol is selected as the wetting agent.
(4) Temperature and time investigation of particle drying
The time and the state of the pellets were examined under different conditions at three temperature ranges of 60 to 69 ℃, 70 to 79 ℃ and 80 to 90 ℃ respectively, and the results are shown in table 23. When the drying temperature is higher than 80 ℃, the particle state is changed, so that the product is determined to be 70-79 ℃ from the two aspects of sample state and production period, and the drying time is 2-4 hours.
Table 23 examination results of drying temperature and time
(5) Determination of particle index
(1) Measurement of the angle of repose
Taking a proper amount of substance reference, adding dextrin, starch and mannitol according to a medicine auxiliary ratio of 1:1:0.5:0.5, uniformly mixing, then spraying 90% ethanol, uniformly stirring, preparing soft materials, granulating, drying to constant weight, and measuring the repose angle. Repeating the operation 3 times generally requires that the repose angle of the granule filler should be controlled within 35 deg. and the results are shown in table 24.
TABLE 24 determination of the angle of repose of particles
(2) Determination of hygroscopicity-Critical relative humidity
1g of the granules dried to a constant weight was taken, placed in a constant weight weighing bottle, precisely weighed, placed in a desiccator containing supersaturated solutions of different concentrations of salt at an opening, placed for 12 hours, weighed, and the results of calculating the percentage of moisture absorption are shown in Table 25. The graph of the relative humidity and the percentage of moisture absorption on the ordinate is shown in FIG. 2, and the tangential line of the graph is plotted, and the critical relative humidity is the abscissa corresponding to the intersection point of the tangential lines.
TABLE 25 Critical relative humidity measurement results
As can be seen from Table 25 and FIG. 2, the critical relative humidity of the two-meter decocted granules is greater than 69%, so that the humidity of the granule production and storage environment should be below 69%.
3. Confirmation of two-instrument granule decocting process study
The preparation process of the granule mainly holds the principle of 'holding to form a cluster and touching to form a powder', obtains 5 key indexes in granule forming by literature investigation, namely granularity, bulk density, repose angle, dissolubility and moisture absorption rate, and sequentially gives objective weight coefficients 15, 20 and 35, and evaluates each factor by taking the indexes as mixed indexes. By combining with experimental study of Critical Relative Humidity (CRH), the humidity requirement of the two-meter decoction granules in actual production is not higher than 69%.
According to the research, the optimal preparation process of the granule is determined by weighing the prepared rehmannia root and the dogwood according to the prescription amount, adding 8 times of water into the prepared rehmannia root and the dogwood according to the prescription amount, decocting for 2 times, each time for 1.5 hours, filtering, concentrating and freeze-drying. Mixing the two-instrument decocted freeze-dried powder according to the proportion of dextrin to starch to mannitol=1:1:0.5:0.5, adding a proper amount of 90% ethanol water solution as a wetting agent to prepare a soft material of 'hand-held agglomeration and instant powder', granulating through a 10-mesh sieve, drying at 70 ℃, and finishing granules to obtain the two-instrument decocted granules.
3. Quality standard
1. Shape: the color, smell and taste of the two-instrument decoction particles are examined, and the two-instrument decoction particles are brown particles, have slight smell and slightly sweet taste.
2. Checking: the preparation is granule, and all examination items are examined under 0104 items in the general rule of the 2020 edition of Chinese pharmacopoeia. The requirements of the granule items, such as appearance, granularity, moisture, dissolubility, loading difference and the like, all meet the regulations.
(1) Appearance inspection
According to the requirements of the granule: the granule should be dried, has uniform granule and uniform color, and has no phenomena of moisture absorption, softening, caking, deliquescence, etc.
(2) Particle size
The percentage of the weight is measured under general rule 0982 (second method double screening method) in the edition 2020 of Chinese pharmacopoeia, and the calculated weight is less than 15%, and the result is shown in Table 26.
Table 26 results of particle size examination
(3) Moisture content
The water content (%) in the test sample is calculated according to the rule 0832 (second method drying method) in the edition 2020 of Chinese pharmacopoeia, and the result is shown in Table 27, and the water content is not more than 8% and meets the regulations.
TABLE 27 results of particle moisture examination
(4) Solubility of
The inspection method of soluble particles under the rule 0104 solubility regulation item in the edition 2020 of Chinese pharmacopoeia shows that 3 batches of samples are slightly turbid and meet the rule.
(5) Difference in loading
The result of the inspection method for the loading difference of the 0101 items in the general rule in the 2020 edition of Chinese pharmacopoeia is shown in table 28, and meets the regulations.
TABLE 28 load variation results
(6) Extract of plant
The content (%) of the alcohol-soluble extract in the test sample was calculated by the hot dipping method under general rule 2201 in the edition 2020 of chinese pharmacopoeia, and the measured results are shown in table 29, and 80% of the tentative alcohol-soluble extract should be not less than 25.0% according to the average measurement result.
TABLE 29 extract measurement results
3. Content determination
(1) Preparation of the solution
Preparation of test solution: 2.0g of the two-instrument decoction particles are taken, added into a volumetric flask with 10mL of methanol for constant volume, and subjected to ultrasonic treatment for 30min, the weight loss is compensated by the methanol, the mixture is uniformly shaken, and the subsequent filtrate is taken, thus obtaining the medicine.
Preparation of a mixed control solution: taking a proper amount of 5-HMF, protocatechuic acid, morroniside, loganin, swertisin and acteoside reference substances, adding methanol into a volumetric flask with a constant volume of 10mL after dissolving, and obtaining mixed reference substance solutions with the concentration of 52.5 mug/mL, 12.5 mug/mL, 115.0 mug/mL, 67.5 mug/mL, 47.5 mug/mL and 27.5 mug/mL respectively.
Preparation of negative sample solution: 2g of negative sample prepared by removing prepared rehmannia root and dogwood respectively, and preparing a negative sample solution according to a preparation method of a sample solution.
(2) Chromatographic conditions
By using Promosil C 18 (4.6 mm. Times.250 mm,5 μm); acetonitrile (B) -0.15% formic acid aqueous solution (A) is taken as a mobile phase, and gradient elution is carried out (0-20 min, 5-9% B, 20-35 min, 9-14.5% A, 35-50 min,14.5% B-17.5% B, 50-62 min,17.5% B-23% B, 62-67 min,23% B, 67-77 min,23% B-29% B, 77-80 min,29% B-95% B); the flow rate is 0.6mL/min; the detection wavelength is 260nm; column temperature is 25 ℃; the sample injection amount was 10. Mu.L.
(3) Methodology investigation
(1) Specificity test
As shown in FIG. 3, the chromatograms of the white solvent, the mixed reference substance, the test substance and the negative sample solution are measured according to the content measuring method, and the result shows that the negative sample has no interference.
(2) Linear relationship investigation
Precisely sucking a proper amount of mixed reference substance under the "content determination" item, adding methanol to dilute into 7 mass concentrations, analyzing under the "chromatographic condition" item, and recording a chromatogram and a peak area. The peak area (Y) is plotted on the ordinate and the concentration (X) is plotted on the abscissa, and the results are shown in table 30 and fig. 4.
Table 30 results of examining the linear relationship of index components
(3) Precision test
Taking the same sample solution under the 'content measurement' item, precisely sucking 10 mu L, continuously sampling for 6 times according to the chromatographic conditions, and recording the peak area. The peak area mean and RSD values were calculated and the results are shown in table 31. The results showed that the RSD values of the peak areas of the index components were all less than 5.00%. The precision of the instrument is good.
Table 31 results of precision test
(4) Stability test
Precisely sucking the same sample solution, taking appropriate amount of solution after 0h, 2h, 4h, 6h, 12h and 24h, injecting into a liquid chromatograph, and recording chromatograms at different times, wherein the result is shown in table 32, and the RSD values of peak areas of the index components are all less than 5.00%. The solution was shown to be stable over 24 h.
Table 32 stability test results
(5) Repeatability test
Taking 6 parts of two decoction particles of the same batch, preparing a test solution according to the content measurement item, precisely sucking 10 mu L of each 6 parts of the test solution, and carrying out parallel experiments according to the chromatographic conditions under the chromatographic conditions item. The reproducibility of the method was evaluated with a relative standard deviation of the peak areas measured in 6 test pieces. As shown in Table 33, the average contents of the respective components were 0.2649, 0.0107, 1.0940, 0.5233, 0.0855 and 0.0882mg/g, respectively, and the RSD values were less than 5.00%, indicating that the reproducibility of the method was good.
TABLE 33 repeatability test results
(6) Recovery test
As shown in tables 34 to 39, two samples of the same batch of the decoction particles with known content were weighed, ground, 6 parts were taken, each of which was precisely weighed, divided into 3 groups, and the 3 groups of 3 parts of the corresponding 80%, 100% and 120% reference solutions were respectively added into 3 groups of volumetric flasks, and tested in parallel, the peak areas of the corresponding chromatographic peaks were recorded, and the obtained chromatographic peak areas were respectively brought into the linear equation to calculate the content of 6 compounds. The recovery rate calculation formula: recovery = (measured-sample content)/addition x 100%. The result shows that the recovery rate of the reference substance is between 95% and 105%, and the RSD of the recovery rate is less than 2%, so that the method has good recovery rate and meets the requirements.
TABLE 34 recovery of 5-HMF loading
TABLE 35 sample recovery of protocatechuic acid
TABLE 36 sample recovery of morroniside
Table 37 loganin sample recovery
TABLE 38 recovery of swertisin loading
Table 39 acteoside sample recovery
(4) Content measurement results
The contents of 5-HMF, protocatechuic acid, morroniside, loganin, swertiside and acteoside in the three batches of two-meter decoction particles were determined and the results are shown in Table 40.
Table 40 results of three sample content measurements
4. Thin layer authentication
(1) Identification of prepared rehmannia root in two-instrument decoction particles
Decocting the granule with two instruments 3g, adding 80% methanol 50ml, ultrasonic treating with power of 300W and frequency of 40 kHz) for 30 min, filtering, evaporating the filtrate, dissolving the residue with 5ml water, extracting with water saturated n-butanol for 4 times under shaking for 10ml each time, mixing n-butanol solutions, evaporating to dryness, dissolving the residue with 2ml methanol to obtain the final productThe two instruments are used for decocting the test solution of the particles. 1.0g of prepared rehmannia root reference medicinal material is prepared and prepared into a reference medicinal material solution. And adding methanol into the calycosin reference substance to obtain a solution containing 1mg per 1ml, and taking the solution as reference substance solution. Taking 3g of negative sample prepared by removing prepared rehmannia root, and preparing a negative control solution according to a preparation method of a sample solution. According to thin layer chromatography (0502 of general rule of 2020 edition of Chinese pharmacopoeia), absorbing 2 μl of each of control solution and control solution, 5 μl of test solution, 5 μl of negative sample solution, and applying to the same silica gel GF 254 The thin layer plate is developed, taken out and dried by taking ethyl acetate-methanol-formic acid-water (16:0.5:2.5:0.5) as developing agent, sprayed with 10% sulfuric acid ethanol solution, dried, heated to 105 ℃ until spots are clear, and inspected under an ultraviolet lamp (365 nm). As shown in fig. 5a, fluorescent spots of the same color appear on the positions corresponding to the control chromatogram and the control chromatogram, and no negative interference occurs.
(2) Identification of dogwood in two-instrument decoction particles
3g of the two-instrument decoction particles are taken, 30mL of methanol is added, ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) is carried out for 20 minutes, filtration is carried out, the filtrate is evaporated to dryness, and 2mL of methanol is added into residues to dissolve the residues, so that the two-instrument decoction particles are taken as a sample solution of the two-instrument decoction particles. 1.0g of dogwood reference medicine is prepared into a reference medicine solution by the same method. And taking a morroniside reference substance and a loganin reference substance, and respectively adding methanol to prepare solutions with 2mg of each 1ml of the solutions as reference substance solutions. Taking 3g of negative sample prepared by removing dogwood, and preparing a negative control solution according to a preparation method of a sample solution. According to thin layer chromatography (0502 of general rule of 2020 edition of Chinese pharmacopoeia), absorbing 2 μl of each of control solution and control solution, 5 μl of test solution, 5 μl of negative sample solution, and applying to the same silica gel GF 254 Spreading with chloroform-methanol (2:1) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until spots are clear, and inspecting under ultraviolet lamp (254 nm). In FIG. 5b, the sample chromatogram shows fluorescence spots of the same color at the positions corresponding to the control chromatogram and the control chromatogram, and no negative interference.
5. Finger print
(1) Preparation of the solution
Preparation of test solution: 2.0g of the two-instrument decoction particles are taken, added into a volumetric flask with 10mL of methanol for constant volume, and subjected to ultrasonic treatment for 30min, the weight loss is compensated by the methanol, the mixture is uniformly shaken, and the subsequent filtrate is taken, thus obtaining the medicine.
Preparation of a mixed control solution: taking a proper amount of 5-HMF, protocatechuic acid, morroniside, loganin, swertisin and acteoside reference substances, adding methanol into a volumetric flask with a constant volume of 10mL after dissolving, and obtaining mixed reference substance solutions with the concentration of 52.5 mug/mL, 12.5 mug/mL, 115.0 mug/mL, 67.5 mug/mL, 47.5 mug/mL and 27.5 mug/mL respectively.
Preparation of negative sample solution: 2.0g of negative sample prepared by removing prepared rehmannia root (dogwood) respectively is prepared into a negative sample solution according to a preparation method of a sample solution.
(2) Chromatographic conditions
By using Promosil C 18 (4.6 mm. Times.250 mm,5 μm); acetonitrile (B) -0.15% formic acid aqueous solution (A) is taken as a mobile phase, and gradient elution is carried out (0-20 min, 5-9% B, 20-35 min, 9-14.5% A, 35-50 min,14.5% B-17.5% B, 50-62 min,17.5% B-23% B, 62-67 min,23% B, 67-77 min,23% B-29% B, 77-80 min,29% B-95% B); the flow rate is 0.6mL/min; the detection wavelength is 260nm; column temperature is 25 ℃; the sample injection amount was 10. Mu.L.
(3) Methodology investigation
(1) Specificity test
As shown in FIGS. 3a to 3e, white solvent, test sample, 5-HMF, protocatechuic acid, morroniside, loganin, swertisin, acteoside and acteoside mixed control and negative sample solution chromatograms were measured according to the content measurement method, and the result shows that the negative sample has no interference.
(2) Precision test
As shown in tables 41 and 42, the same sample solution is precisely sucked, the sample is continuously injected for 6 times according to the condition under the 'chromatographic condition', the retention time and the chromatographic peak area of chromatographic peaks of 6 chromatograms are recorded, the retention time of the 12 # chromatographic peak loganin is stable and the separation degree is better, so the 12 # peak is used as a reference peak (S), the relative retention time and the relative peak area RSD value of each common peak are calculated to be less than 5.00%, and the result shows that the instrument is stable and the precision is good
Table 41 precision versus retention time
Table 42 precision versus peak area
(3) Repeatability test
As shown in tables 43 and 44, 6 parts of sample solutions are prepared in parallel by taking the same part of particles, and sample injection is carried out respectively according to the conditions under the 'chromatographic conditions', so that the relative retention time and the RSD value of the relative peak area of each common peak are less than 5.00%, and the result shows that the repeatability of the method is good.
Table 43 repeatability versus retention time
Table 44 repeatability versus peak area
(4) Stability test
As shown in tables 45 and 46, the same sample solution was precisely sucked, and after 0h, 2h, 4h, 6h, 12h and 24h, a proper amount of the solution was injected into a liquid chromatograph, and chromatograms at different times were recorded, and the result showed that the relative retention time of each common peak and the RSD value of the relative peak area were all <5.00%. The solution was shown to be stable over 24 h.
Table 45 stability versus retention time
Table 46 stability versus peak area
(4) Establishment of fingerprint spectrum of two-instrument decoction particles
(1) Common mode
According to literature search, the two-instrument decoction is found to contain 5-HMF, protocatechuic acid, morroniside, loganin, swertiside, acteoside and the like, so that 5-HMF, protocatechuic acid, morroniside, loganin, swertiside and acteoside are selected as reference substances. The chromatographic analysis is carried out on the sample solution of the two-instrument decocted particles, 19 chromatographic peaks are obtained through total separation, and the retention time of the chromatographic peaks is compared with that of a reference substance, so that the result shows that the No. 1 chromatographic peak is 5-HMF, the No. 2 chromatographic peak is protocatechuic acid, the No. 7 chromatographic peak is morroniside, the No. 12 chromatographic peak is loganin, the No. 13 chromatographic peak is swertiside, the No. 16 chromatographic peak is acteoside, and other chromatographic peaks are still to be further attributed.
As shown in fig. 6 and 7, according to the above chromatographic conditions, a sample solution of 10 batches of two-meter decoction particles is subjected to chromatographic analysis, each chromatographic parameter is recorded, and the chromatogram data of the sample solution of 10 batches of two-meter decoction particles is imported into software by using the "traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A edition", S1 is set as a reference spectrum, and a reference fingerprint is generated through multipoint correction and data matching by adopting a median method. And determining 19 common peaks of 10 batches of two-instrument decocted particles according to the matching result, and obtaining the fingerprint of 10 batches of two-instrument decocted particles.
As shown in tables 47 and 48, the relative retention time and the relative peak area of 19 common chromatographic peaks were calculated for 10 samples, using the 12 # chromatographic peak loganin as a reference peak. Relative retention time = retention time of each peak/retention time of reference peak; relative peak area = peak area of each peak/peak area of reference peak.
Table 47 relative retention time of common peaks
Table 48 relative peak area of common peaks
As can be seen from the graph, the relative retention time of 19 common chromatographic peaks in the HPLC fingerprint of 10 batches of the two-instrument decoction particles has smaller RSD value, the RSD value is smaller than 1.00%, the relative peak area of the common peaks has RSD value between 6.57 and 33.51, the peak area difference is larger, which indicates that the two-instrument decoction particles in different batches have the same chemical components, but the component contents in different batches are different.
(2) Similarity evaluation
As shown in table 49, the chromatographic analysis is performed on 10 batches of two-instrument decoction particles according to the above chromatographic conditions, the chromatographic data patterns of 10 batches of two-instrument decoction particles are imported into the system by using the "traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A edition", and a common pattern is established for two-instrument decoction particles in different batches by adopting an angle cosine method, so that the results show that the similarity is above 0.900, the overall chromatograms are consistent, and the similarity is better.
Table 49 similarity of two-batch decoction particles
The preparation process of the granule for decoction by two instruments in the medical prescription provided by the invention adopts a water decoction method for extraction. The total number of factors influencing the extraction process is 3, namely the water adding multiple, the decoction times and the decoction time, and the factors have interaction, so that the experimental workload is large, the implementation is difficult, and the efficient and economic L is selected 9 (3 4 ) The experiments were designed in orthogonal experiments. The prepared rehmannia root in the two-instrument decoction formula takes acteoside as an index component in the pharmacopoeia of 2015 edition, and the dogwood takes morroniside and loganin as index components in the pharmacopoeia of 2020 edition, so that the contents of acteoside, morroniside and loganin are jointly taken as investigation indexes of an extraction process, and whether the extraction of the two-instrument decoction effective components is sufficient or not can be further described. The optimal process of two-instrument decoction is to add 8 times of water for 2 times, each time is 1.5 hours, and the contents and the ointment yield of the acteoside, the morroniside and the loganin are stable in three batches of verification tests, which proves that the extraction process is reasonable, reliable and stable, and the two-instrument decoction can play the maximum clinical curative effect from the angles of the acteoside content, the morroniside content and the loganin content.
Based on the screened optimal extraction process, the two-instrument freeze-dried powder is prepared, and the preparation is carried out in the early stage for the forming process of the granules. The preparation process of the granule mainly holds the principle of 'holding to form a cluster and touching to form a powder', obtains 5 key indexes in granule forming by literature investigation, namely granularity, bulk density, repose angle, dissolubility and moisture absorption rate, and sequentially gives objective weight coefficients 15, 20 and 35, and evaluates each factor by taking the indexes as mixed indexes. By combining with experimental study of Critical Relative Humidity (CRH), the humidity requirement of the two-meter decoction granules in actual production is not higher than 69%. According to the research, the optimal preparation process of the granule is that the medicine powder, namely dextrin and starch, is mixed uniformly in the proportion of mannitol=1:1:0.5:0.5, and a proper amount of 90% ethanol water solution is added as a wetting agent to prepare a soft material which is 'held by hands, agglomerated and scattered immediately after touching', is granulated by a 10-mesh sieve, dried at 70 ℃, and granulated, thus obtaining the two-instrument decoction granule.
The invention establishes the quality standard of the two-instrument decoction granules. The content measurement, the specificity experiment and the methodology investigation are carried out on the 5-HMF, the protocatechuic acid, the morroniside, the loganin and the acteoside in the two-instrument decoction particles by utilizing the HPLC content simultaneous measurement technology, so that the stability of the results is strong, the repeatability is good and the accuracy is high; finally, according to the requirements of the general rule 0104 granule item in the edition 2020 of Chinese pharmacopoeia, the granularity, the moisture, the dissolubility, the loading difference and the extract of three batches of samples are checked, and the results all meet the regulations.
The two-instrument decoction particles are composed of two medicines of prepared rehmannia root and dogwood, have more chemical components and have certain interference with each other, and according to the related guiding principle of the particles, the invention performs thin-layer chromatography identification on each medicine in the prescription based on the identification and determination methods of each medicine in the prescription defined in Chinese pharmacopoeia, and optimizes the method to obtain the thin-layer chromatography condition with clear spots and no negative interference.
The invention establishes the fingerprint spectrum of the two-instrument decoction particles and comprehensively evaluates the quality. The similarity of the fingerprints established by the invention is greater than 0.94, the fingerprints are attributed, 19 common peaks are determined, and the fingerprints are respectively attributed to prepared rehmannia root and dogwood. The fingerprint established by the invention can comprehensively reflect the whole picture, and provides reference for the subsequent study of the new medicine of the two-instrument decocted granules.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims (10)

1. The preparation process of the decoction granules by two instruments towards the medical prescription is characterized by comprising the following steps:
extracting radix rehmanniae Preparata and Corni fructus with water, concentrating, and lyophilizing to obtain two-instrument decoction lyophilized powder;
mixing the two-instrument decocted freeze-dried powder and auxiliary materials uniformly, adding a wetting agent, preparing primary product particles through a sieve, and drying to obtain the two-instrument decocted particles;
wherein, the mass ratio of the prepared rehmannia root to the dogwood is 2:1, the mass ratio of the two-instrument decocted freeze-dried powder to the auxiliary materials is 1:2, the auxiliary materials comprise dextrin, starch and mannitol.
2. The process for preparing the granules decocted in two instruments towards the medical prescription as claimed in claim 1, wherein the water adding extraction process comprises the following steps: adding 6-10 times of water into the medicinal materials, and decocting for 1-3 times, wherein each time of decoction lasts for 0.5-1.5 hours.
3. The process for preparing the two-instrument decoction granules towards the medical prescription according to claim 1 or 2, wherein the mass ratio of the two-instrument decoction lyophilized powder to the dextrin to the starch to the mannitol is as follows: 1:0.5 to 1:0.25 to 1:0.25 to 0.75.
4. The process for preparing the granules decocted in two instruments towards the medical side as claimed in claim 3, wherein the wetting agent is an ethanol solution with the concentration of 80% -95%.
5. The process for preparing the granules decocted in two instruments towards the medical prescription as claimed in claim 3, wherein the drying temperature after preparing the primary granules is 60 ℃ to 69 ℃,70 ℃ to 79 ℃ or 80 ℃ to 89 ℃; and the drying time is 2-4 h.
6. The preparation process of the two-instrument decoction granules towards the medical prescription as claimed in claim 4 or 5, wherein the prepared rehmannia root and the dogwood are taken, 8 times of water is added, the decoction is carried out for 2 times, each time of decoction is carried out for 1.5 hours, the concentration is carried out after filtration, and the two-instrument decoction freeze-dried powder is obtained after freeze drying;
the two instruments are decocted to freeze-dried powder, dextrin, starch and mannitol according to the mass ratio of 1:1:0.5: and (3) after uniformly mixing 0.5, adding an ethanol solution with the concentration of 90%, granulating by a 10-mesh sieve, and drying at 70 ℃ to obtain the two-instrument decoction granules.
7. The method for detecting the quality standard of the decoction granules by two instruments towards the medical prescription is characterized by comprising the steps of property detection, appearance detection, granularity detection, moisture detection, solubility detection, content difference detection, extract detection, thin-layer chromatography detection, fingerprint detection and content detection;
wherein, the content measurement process comprises:
preparing a test solution: adding methanol into the two decoction particles to fix volume, performing ultrasound, supplementing the weight loss with methanol, shaking uniformly, filtering, and collecting the subsequent filtrate to obtain the sample solution;
Preparing a mixed reference substance solution: taking a proper amount of 5-HMF, protocatechuic acid, morroniside, loganin, swertisin and acteoside as reference substances, adding methanol for dissolving, and then fixing the volume to obtain mixed reference substance solutions with the concentration of 52.5 mug/mL, 12.5 mug/mL, 115.0 mug/mL, 67.5 mug/mL, 47.5 mug/mL and 27.5 mug/mL respectively;
preparing a negative sample solution: respectively taking negative samples prepared by removing radix rehmanniae Preparata and Corni fructus, adding methanol to constant volume, performing ultrasound, supplementing reduced weight with methanol, shaking, filtering, and collecting subsequent filtrate to obtain the negative sample solution;
and respectively precisely sucking the sample solution, the mixed reference substance solution and the negative sample solution, injecting the sample solution into a high performance liquid chromatograph, and measuring by gradient elution to obtain the content.
8. The method for detecting the quality standard of the granules decocted in two instruments towards the doctor according to claim 7, wherein the chromatographic conditions are as follows: using chromatographic columns as Promosil C 18 Is used as a stationary phase, the column temperature is 25 ℃, the detection wavelength is 260nm, the flow rate is 0.6mL/min, the analysis time is 80min, and 10 mu L of sample injection amount is adopted for injection into a high performance liquid chromatograph; and
the elution procedure for the gradient elution was as follows:
At 0 min, mobile phase A was 95% formic acid in water and mobile phase B was 5% acetonitrile;
at 20 minutes, mobile phase A was 91% formic acid in water and mobile phase B was 9% acetonitrile;
at 35 minutes, mobile phase A was 85.5% formic acid in water and mobile phase B was 14.5% acetonitrile;
at 50 minutes, mobile phase A was 82.5% formic acid in water and mobile phase B was 17.5% acetonitrile;
at 62 minutes, mobile phase A was 77% formic acid in water and mobile phase B was 23% acetonitrile;
at 67 minutes, mobile phase A was 77% formic acid in water and mobile phase B was 23% acetonitrile;
at 77 minutes, mobile phase A was 71% formic acid in water and mobile phase B was 29% acetonitrile;
at 80 minutes, mobile phase A was 5% formic acid in water and mobile phase B was 95% acetonitrile;
the column temperature is 25 ℃; flow rate: 0.6mL/min; sample injection amount: 10 mu L.
9. The method for detecting the quality standard of the two-instrument decoction granules towards the medical side according to claim 8, wherein the fingerprint spectrum in the fingerprint spectrum identification has 19 common fingerprint peaks, and the relative retention time is respectively:
peak No. 1: 0.368-0.370; peak No. 2: 0.512-0.514; peak No. 3: 0.553 to 0.556; peak No. 4: 0.609 to 0.613; peak No. 5: 0.657 to 0.662; peak No. 6: 0.710 to 0.712; peak No. 7: 0.759 to 0.760; peak No. 8: 0.830 to 0.832; peak No. 9: 0.854 to 0.857; peak No. 10: 0.875 to 0.879; peak No. 11: 0.920 to 0.923; peak No. 12 is a reference peak: 1.000; peak No. 13: 1.021 to 1.022; peak No. 14: 1.107 to 1.108; peak No. 15: 1.265 to 1.266; peak No. 16: 1.469 to 1.471; peak No. 17: 1.526 to 1.531; peak No. 18: 1.570-1.573; peak No. 19: 1.609 to 1.613.
10. The method for detecting the quality standard of the two-instrument decoction granules towards the medical side according to claim 7, wherein the thin-layer chromatography identification process comprises the following steps:
adding 80% methanol into the two-instrument decoction particles, filtering, evaporating filtrate to dryness, dissolving residues in water, shaking and extracting with water saturated n-butanol, evaporating to dryness, dissolving residues in methanol, and taking the residues as a sample solution of the two-instrument decoction particles; preparing reference medicinal material solution from radix rehmanniae Preparata by the method for preparing sample solution; adding methanol as reference solution; taking a negative sample prepared by removing prepared rehmannia root, and preparing a negative control solution according to a preparation method of a sample solution; sucking 2 μl of each of the control solution and the control solution by thin layer chromatography, 5 μl of the test solution and 5 μl of the negative sample solution, and spotting on the same silica gel GF 254 Spreading on a thin layer plate with ethyl acetate-methanol-formic acid-water as developing agent at a ratio of 16:0.5:2.5:0.5, taking out, air drying, spraying 10% sulfuric acid ethanol solution, air drying, heating at 105deg.C until spots are clear, and inspecting under 365nm ultraviolet lamp; in the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control sample, and no negative interference exists.
Taking two-instrument decoction particles, adding methanol, filtering, evaporating filtrate to dryness, and adding methanol into residues to dissolve the residues to obtain a sample solution of the two-instrument decoction particles; preparing a control medicinal material solution from a dogwood control medicinal material according to a preparation method of a sample solution; taking a morroniside reference substance and a loganin reference substance, and respectively adding methanol to prepare reference substance solutions; taking a negative sample prepared by removing dogwood, and preparing a negative control solution according to a preparation method of a sample solution; sucking 2 μl of each of the control solution and the control solution by thin layer chromatography, and dissolving negative sample with 5 μl of the test solutionLiquid 5. Mu.L, respectively spotted on the same silica gel GF 254 Spreading chloroform-methanol as developing agent at a ratio of 2:1, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until spots are clear, and inspecting under 254nm ultraviolet lamp; in the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control sample, and no negative interference exists.
CN202311720162.8A 2023-12-14 2023-12-14 Preparation process of medicinal prescription two-instrument decoction granules and quality standard detection method Pending CN117695232A (en)

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