A Buzhong Yiqi Decoction granule, its preparation method, content, and thin-layer identification method

Abstract

The present invention belongs to the technical field of the preparation of traditional Chinese medicine preparations, and particularly relates to a Buzhong Yiqi Decoction granule, its preparation method, content, and thin-layer identification method; the granule prepared by the present invention has rapid drug absorption, high bioavailability, good stability, convenient taking, carrying and storage, accurate and easy-to-control dosage, and a simple production process and economical cost. The granule is prepared by the fluidized bed granulation method, and it is verified that the formability is good, and the granule preparation process is stable and feasible, which can lay a solid foundation for the subsequent research and development of enterprises. The thin-layer identification method adopted has good durability and accurate identification. And it is determined that the content of tangerine peel in this product calculated as hesperidin (C 28 H 34 O 15 ) should not be less than 2.72 mg / g, and the content of roasted licorice calculated as glycyrrhizic acid (C 42 H 62 O 16 ) should not be less than 1.93 mg / g.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of traditional Chinese medicine preparations, and particularly relates to a Buzhong Yiqi Decoction granule, its preparation method, content, and thin-layer identification method. Background Art

[0002] The prescription of Buzhong Yiqi Decoction comes from "Volume 3 of 'Differentiation and Treatment of Internal and External Injuries'", and it is one of the representative prescriptions for replenishing qi. Functional indications: replenishing middle qi and benefiting qi, ascending yang and lifting prolapse, used for diarrhea, rectal prolapse, and uterine prolapse caused by weakness of the spleen and stomach and sinking of middle qi, with symptoms such as fatigue, reduced appetite, abdominal distension, loose stools with long-term diarrhea, anal prolapse or rectal prolapse, and uterine prolapse.

[0003] The existing patent document with the publication number of CN102293995B discloses that the Buzhong Yiqi Granules include: raw materials and excipients with a mass ratio of (4:1)-(6:1), where: the raw materials are composed of the following components in parts by mass: roasted Astragalus membranaceus 18-22 parts; Codonopsis pilosula 5-6.5 parts; roasted Glycyrrhiza uralensis 9-11 parts; Angelica sinensis 5-6.5 parts; stir-fried Atractylodes macrocephala 5-6.5 parts; Cimicifuga foetida 5-6.5 parts; Bupleurum chinense 5-6.5 parts; Pericarpium Citri Reticulatae Viride 5-6.5 parts; Zingiber officinale 1.5-2.5 parts; Ziziphus jujuba 3.5-4.5 parts; the excipients are composed of lactose and dextrin, lactose accounts for 50%-90% of the total mass of the excipients, and the rest is dextrin; or lactose is used to replace lactose and dextrin in the excipients.

[0004] However, the existing technology has not further studied the contents of effective drug components such as hesperidin and glycyrrhizic acid in Buzhong Yiqi Decoction, and it is not clear whether such processes and ratios have an impact on the effective components of the drug. For traditional Chinese medicine preparations, the identification and determination of their effective components are very important processes in the modernization of traditional Chinese medicine. Therefore, the present invention mainly screens and optimizes the preparation process, prescription, etc. of Buzhong Yiqi Decoction to provide a Buzhong Yiqi Decoction granule with clear drug efficacy and available for reference in medication. Summary of the Invention

[0005] The present invention provides a Buzhong Yiqi Decoction granule and its preparation method to solve the above problems.

[0006] Specifically, it is achieved through the following technical solutions:

[0007] 1. A Buzhong Yiqi Decoction granule, the prescription of the Buzhong Yiqi Decoction granule is: roasted Astragalus membranaceus 650-670 g, Codonopsis pilosula 185-213 g, roasted Glycyrrhiza uralensis 310-337 g, stir-fried Atractylodes macrocephala with bran 185-213 g, Angelica sinensis 185-213 g, Cimicifuga foetida 185-213 g, Bupleurum chinense 185-213 g, Pericarpium Citri Reticulatae Viride 185-213 g, Zingiber officinale 60-72 g, Ziziphus jujuba 130-135 g.

[0008] 2. The preparation method of the above Buzhong Yiqi Decoction granule, including the following steps:

[0009] (1) Soak 10 kinds of medicinal materials in water for 0.5 h and then decoct them three times, each time for 2 h, and combine the decoctions of the three times; the water addition amount for the first decoction is 10 times the mass of all the medicinal materials, and the water addition amounts for the second and third times are both 8 times the mass of all the medicinal materials.

[0010] (2) Filter the combined decoction, concentrate the filtrate into a clear paste, and then mix it with 450 - 570 g of auxiliary materials to prepare granules by the boiling granulation method.

[0011] Furthermore, the relative density of the clear paste is 1.10 - 1.15.

[0012] Furthermore, the auxiliary materials are composed of maltodextrin and soluble starch in a mass ratio of 1:3.

[0013] Furthermore, the content of hesperidin in the granules is not less than 2.72 mg / g, and the content of glycyrrhizic acid is not less than 1.93 mg / g.

[0014] I. Related screening research experiments on Buzhong Yiqi Decoction granules

[0015] 1. Screening and optimization confirmation of extraction orthogonal experiment

[0016] 1.1 Examine the three main factors affecting the decoction effect, namely water addition amount, decoction time, and decoction times. According to 3 levels for each factor, use the L 9 (3 4 ) orthogonal table to arrange the experiment, and the results are shown in Table 1. The water decoction process is screened by orthogonal experiment. In order to enable the process to fully reflect the drug efficacy, and combined with the characteristics of the drug composition of this formula and the functions and indications of this formula, the decoction rate and the transfer rate of the index components are used to evaluate the process, and the water extraction conditions are optimized. The test results are recorded in Tables 2 and 3.

[0017] Table 1 L 9 (3 4 ) Orthogonal experiment factor - level table

[0018]

[0019] Table 2 L 9 (3 4 ) Orthogonal experiment table

[0020]

[0021] Note: Comprehensive score = transfer rate of hesperidin in each sample / maximum transfer rate of hesperidin in this group * 0.3333 * 100% +

[0022] transfer rate of glycyrrhizic acid in each sample / maximum transfer rate of glycyrrhizic acid in this group * 0.3333 * 100% +

[0023] The extraction rate of each sample / the maximum extraction rate in the group * 0.3333 * 100%

[0024] Table 3L 9 (3 4 ) ANOVA results of the comprehensive score of the orthogonal experiment

[0025]

[0026] 1.2 Conclusion

[0027] Through intuitive analysis, the optimal extraction process is A 3 B 1 C 3 D 2 ; Combining the results of the ANOVA of the comprehensive score, only the number of extraction times has a significant effect on the extraction process (P < 0.05), and the other investigated factors have no significant effect on the extraction process (P > 0.05); Combining with the production process requirements and the range analysis of the extraction rate and the transfer rate of the index components, the optimal extraction process combination is determined as: A 3 B 1 C 3 D 2 , that is, extract three times, add 10 times water for the first time, soak for 60 minutes, extract for 120 minutes after boiling, add 8 times water for the second time, extract for 120 minutes after boiling, add 8 times water for the third time, extract for 120 minutes after boiling, and filter the three decoctions with a 200-mesh filter cloth.

[0028] 1.3 Optimization and confirmation of the screening process conditions

[0029] To further confirm the feasibility of the extraction process combination screened by the orthogonal experiment, comparative studies on optimization and confirmation were carried out again under the following different process combination conditions, and the research results are shown in Table 4.

[0030] ① Extraction process combination (A3B1C3D2) initially screened by the orthogonal experiment: Extract three times, add 10 times water for the first time, soak for 60 minutes, extract for 120 minutes after boiling, add 8 times water for the second time, extract for 120 minutes after boiling, add 8 times water for the third time, extract for 120 minutes after boiling, filter the three decoctions with a 200-mesh filter cloth, and finally combine the decoctions (batch number: 210818).

[0031] ② Process combination (A3B1C3D1) determined by investigating the soaking time: Extract three times, add 10 times water for the first time, soak for 30 minutes, extract for 120 minutes after boiling, add 8 times water for the second time, extract for 120 minutes after boiling, add 8 times water for the third time, extract for 120 minutes after boiling, filter the three decoctions with a 200-mesh filter cloth, and finally combine the decoctions (batch number: 210825-1).

[0032] ③ Extraction times comparison process combination (A2B1C3D1): Extract twice. For the first time, add 10 times the amount of water, soak for 30 minutes, and extract for 120 minutes after boiling. For the second time, add 8 times the amount of water and extract for 60 minutes after boiling. Filter the decoctions of both times with a 200-mesh filter cloth, and finally combine the decoctions (batch number: 210825-2). The results are shown in the following table.

[0033] Table 4 Optimization and confirmation results of the screening process conditions

[0034]

[0035] Conclusion: It can be seen from the results that among the three combinations, the combination (A3B1C3D1) has the highest paste yield and transfer rate. Therefore, the best extraction process can be confirmed as the combination (A3B1C3D1): Extract three times. For the first time, add 10 times the amount of water, soak for 30 minutes, and extract for 120 minutes after boiling. For the second time, add 8 times the amount of water and extract for 120 minutes after boiling. For the third time, add 8 times the amount of water and extract for 120 minutes after boiling. Filter the decoctions of all three times with a 200-mesh filter cloth.

[0036] Through the orthogonal experiment screening investigation of the extraction process and the optimization and confirmation research of the screening process conditions, the small-scale extraction process of Buzhong Yiqi Decoction Granules was determined as (A3B1C3D1): That is, extract three times. For the first time, add 10 times the amount of water, soak for 30 minutes, and extract for 120 minutes after boiling. For the second time, add 8 times the amount of water and extract for 120 minutes after boiling. For the third time, add 8 times the amount of water and extract for 120 minutes after boiling. Filter the decoctions of all three times with a 200-mesh filter cloth.

[0037] 2. Concentration process investigation

[0038] 2.1 Combine and mix the orthogonal decoctions. Take 6 portions of 3000 g of the combined decoctions respectively, and concentrate them to the same volume under reduced pressure using the same rotary evaporator at different temperatures. Compare the effects of different concentration temperatures on the concentrated solution. Using the paste yield of the clear paste and the transfer rate of the index components as evaluation indicators, optimize the best concentration temperature and evaluate the concentration process. The results are shown in Table 5.

[0039] Table 5 Investigation results of concentration temperature

[0040]

[0041] By using the same instrument and the same batch of samples with the same amount for the concentration process investigation and comparing the effects of different concentration temperatures on the paste yield and index components, the results show that the paste yield and the transfer rate of the index components are the highest at 70 °C. Initially, 70 °C is determined as the best concentration temperature. And at 65 °C and 75 °C, the paste yield and the transfer rate of the index components do not differ significantly from those at 70 °C. It is confirmed that the fluctuation range of the concentration temperature is 70 + ± 5 °C.

[0042] 2.1 Extraction and Concentration Process Validation and Confirmation

[0043] According to the extraction and concentration process initially confirmed by the research, conduct the small-scale process validation of extraction and concentration, and simultaneously conduct a comparative study with the clinical standard decoction to further confirm the feasibility of the small-scale extraction and concentration process.

[0044] 2.1.1 Preparation of Three Batches of Small-Scale Samples

[0045] Weigh 3 batches of clinical prescription cut crude drugs, with 3 doses for each batch, and extract and concentrate them according to the selected optimal extraction process conditions. Record the volume of the medicinal liquid for each batch, and measure the relative density of the medicinal liquid. The experimental results are recorded in Table 6.

[0046] Table 6 Records of the Preparation of Three Batches of Small-Scale Validation

[0047]

[0048] Based on the hot and cold measurement results of the relative density of the concentrated paste obtained from the three batches of small-scale validation, preliminarily determine that the relative density of the clear paste obtained by concentration is: 1.10 - 1.15 (55°C - 65°C)

[0049] 2.1.2 Determination of the Yield of Extract and the Transfer Rate of Index Components for Three Batches of Small-Scale Samples and Three Batches of Standard Decoctions

[0050] Table 7 Results of the Yield of Extract and the Transfer Rate of Index Components for Three Batches of Small-Scale Samples and Three Batches of Standard Decoctions

[0051]

[0052] Conclusion: The results of the small-scale process validation of the extraction process show that the yields of extract and the transfer rates of index components of the three batches of products are basically the same, indicating that the confirmed small-scale extraction process has good reproducibility and the small-scale process is stable and controllable. The concentration process shows that the yields of extract and the transfer rates of index components of the three batches of products slightly decrease, but are all within the controllable range, with good reproducibility and relatively stable concentration process. Compared with the three batches of clinical standard decoction samples, the yields of extract and the transfer rates of index components of the three batches of extraction and concentration samples are significantly better than those of the clinical standard decoction samples, with good process reproducibility, stable and controllable process, indicating that the determined small-scale extraction and concentration process is stable and feasible.

[0053] 3. Confirmation of the Screening of the Formulation Prescription and Investigation of the Molding Process

[0054] After weighing and extracting and concentrating 300 doses of clinical prescription cut crude drugs, conduct the investigation of the molding process of granules and the screening of the formulation prescription using their extract. The corresponding parameters of the extract (batch number: 211015) are shown in Table 8.

[0055] Table 8 Extract Parameters

[0056]

[0057] 3.1 Investigation on the Forming Process of the Preparation

[0058] Since this is a classical famous prescription with a relatively small prescribed dose, and considering the results of the extract yield detection in the early stage, which meet the requirements of the daily dosage of the granule, the dosage form is determined to be granules. In this screening, three common granulation methods are mainly compared and investigated, namely wet granulation (atmospheric drying), fluidized bed granulation (fluidized bed drying), and dry granulation (spray-dried dry extract powder plus excipients). Weigh 40 doses of the extract respectively and conduct investigations on the three granulation methods. By comparing the granulation process of the granules, the granule forming situation, the carrying capacity of the active ingredients in the granules, and the dissolution of the granules, etc., the best granulation and forming process is screened out. To ensure the comparability of the experiments, the types of excipients used in the granulation process are the same. The experimental results are shown in Table 9.

[0059] Table 9 Results of Different Granulation Methods

[0060]

[0061]

[0062] From the comparison results in the above table, it can be seen that fluidized bed granulation has the highest yield, the best granule forming, and the largest transfer rate of active ingredients. Considering factors such as actual production and energy consumption, fluidized bed granulation (fluidized bed drying) is selected as the granule forming process for Buzhong Yiqi Decoction granules.

[0063] 3.2 Screening of the Types of Excipients

[0064] Selection of the types of excipients: Medicinal excipients are the general term for other components in the preparation except the active ingredients, and their main functions in the preparation include excipients, stabilizers, and improving compliance, etc. Since Chinese medicine water extracts and clear extracts are rich in Chinese medicine components such as polysaccharides, tannins, and mucilages, etc., sticky wall problems are likely to occur during fluidized bed granulation. In addition to improving the equipment performance, the ideal method to prevent sticky wall is to add appropriate excipients that are effective for various sticky substances as fillers to promote the drying of the extraction liquid and Chinese medicine clear extract. Combining the quality requirements of the dissolution of the granules, the compliance of the drug, and the needs of the applicable population, maltodextrin and soluble starch with low sweetness, low calorie, high nutrition, good solubility, strong heat resistance, low hygroscopicity, not easy to agglomerate, and economic and safe are preferentially selected as the common excipients during fluidized bed granulation in this experiment.

[0065] 3.3 Screening of the Dosage of Excipients

[0066] Referring to the preparation process requirements of different granulation methods for the production of traditional Chinese medicine granules, the dosage of excipients used in the research was initially investigated. The granule yield, drying state, and ease of drying were used as evaluation indicators for preliminary evaluation. The specific method was to take the above 40-dose extracts, each weighing 3.0 kg, select reasonable fluidized granulation process parameters according to the design scheme in the following table, and conduct an investigation test on the addition amount of excipients for fluidized granulation. The test results are shown in Table 10.

[0067] Table 10 Screening Results of Excipient Dosage

[0068]

[0069] From the results of the above table, it can be seen that when 300 g of excipients were added, there were wall sticking and caking phenomena, and it was not easy to dry; when 360 g of excipients were added, there was a slight wall sticking phenomenon; when 480 g or 560 g of excipients were added, there was no wall sticking phenomenon, it was easy to dry, the drying effect was good, the granule formability was good and the yield was high, but when 560 g of excipients were added, the daily dosage was relatively large after conversion. Therefore, it was most appropriate to choose to add 480 g of excipients.

[0070] 4. Investigation of Granulation Methods

[0071] Table 11 Determination Results of Hesperidin in Investigation of Granulation Methods

[0072]

[0073]

[0074] Table 12 Determination Results of Glycyrrhizic Acid in Investigation of Granulation Methods

[0075]

[0076] The test results showed that among the three granulation methods, the content of hesperidin was in the order of wet granulation < dry granulation < fluidized granulation, and the content of glycyrrhizic acid was in the order of wet granulation < dry granulation < fluidized granulation. The test results indicated that choosing fluidized granulation, the content of each component was the highest.

[0077] 5. Pilot Study on Buzhong Yiqi Decoction Granules

[0078] According to the confirmed extraction, concentration, and forming processes, three batches of pilot samples were prepared for process verification. Production was scaled up by 200 times according to the proposed preparation process conditions, and the pilot scale-up production plan for the preparation process of Buzhong Yiqi Decoction granules was initially determined, as shown in Table 13. The production process indicators were comprehensively evaluated, and the quality of the decoction, concentrated solution, and finished product was evaluated to investigate the rationality of the preparation process of this product.

[0079] Table 13 Pilot Scale-up Production Plan for the Preparation Process of Buzhong Yiqi Decoction Formula Granules

[0080]

[0081] According to the above table plan, a batch of pilot-scale experiments were carried out to further confirm the production process of Buzhong Yiqi Decoction formula granules. The specific operating parameters are shown in Table 13, and the data are shown in Tables 14 and 15.

[0082] Table 14 Experimental result data of pilot-scale production in the preparation process

[0083]

[0084] Table 15 Content results of Buzhong Yiqi Decoction formula granules

[0085]

[0086] 5.1 Preparation of three batches of pilot-scale samples

[0087] Weigh 200 doses of clinical prescription cut herbs in three batches respectively, and carry out extraction, concentration and granulation according to the technological conditions in Table 13. The experimental results are recorded in Table 16.

[0088] Table 16 Verification preparation records of three batches of pilot-scale experiments

[0089]

[0090]

[0091] 5.2 Results of determination of extract yield, transfer rate of index components and content in three batches of pilot-scale experiments

[0092] Table 17 Results of extract yield and transfer rate of index components in three batches of pilot-scale experiments

[0093]

[0094] Table 18 Content results of three batches of pilot-scale granules

[0095]

[0096] Conclusion: The extract yield, transfer rate and granule content of the three batches of pilot-scale samples tend to be stable, indicating the feasibility of this process.

[0097] The pilot-scale samples were analyzed and determined by liquid chromatography. The results showed that the transfer rate of hesperidin in the decoction of the pilot-scale verification was 56%, and the transfer rate of the three batches of pilot-scale decoctions was 57%; the transfer rate of hesperidin in the concentrated decoction of the pilot-scale verification was 52%, and the transfer rate of the three batches of pilot-scale concentrated decoctions was 51%; the content of hesperidin in the pilot-scale verification granules was 3.80 mg / g, and the content of hesperidin in the three batches of pilot-scale granules was 3.85 mg / g. The results showed that there was no significant difference in the content of hesperidin in the pilot-scale verification.

[0098] The transfer rate of glycyrrhizic acid in the decoction was verified to be 71% in the pilot test, and the transfer rates of the three batches of pilot decoctions were 73%; the transfer rate of glycyrrhizic acid in the concentrated solution was verified to be 68% in the pilot test, and the transfer rates of the three batches of pilot concentrated solutions were 70%; the content of glycyrrhizic acid in the granules was verified to be 2.7 mg / g in the pilot test, and the contents of glycyrrhizic acid in the three batches of pilot granules were 2.8 mg / g. The results showed that there was no significant difference in the content of glycyrrhizic acid in the pilot test.

[0099] 6. TLC Identification Research

[0100] 6.1 Materials and Reagents

[0101] Silica gel G TLC plates (10 cm × 10 cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20200601), silica gel G TLC plates (10 cm × 10 cm, Sinopharm Chemical Reagent Co., Ltd., batch number: 20200110), silica gel G TLC plates (10 cm × 10 cm, Merck KGaA), silica gel GF254 TLC plates (10 cm × 10 cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20200601), mL204 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.).

[0102] Buzhong Yiqi Decoction formula granules (211015), samples lacking roasted Astragali Radix (211101-1), samples lacking Cimicifugae Rhizoma (211101-3).

[0103] 6.2 TLC Identification Method

[0104] 6.2.1 TLC Identification of Roasted Astragali Radix

[0105] Take 0.5 g of this product, add 10 mL of methanol solution, ultrasonically treat for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution. Separately, take 1 g of the control crude drug of roasted Astragali Radix, add 25 mL of distilled water, reflux for 1 h, cool, filter, take the filtrate and evaporate to dryness. Dissolve the residue in 5 mL of methanol to obtain the control crude drug solution. According to the TLC method (General Rule 0502), absorb 8 μL of each of the above solutions and spot them on the same GF 254 silica gel plate, use chloroform-methanol (10:1) as the developing solvent, develop, take out, dry in air, and examine under ultraviolet light (254 nm). In the chromatogram of the test solution, at the corresponding position to the chromatogram of the control crude drug, fluorescent spots of the same color appear.

[0106] Conclusion: As can be seen from the figure, spots of the same color appear at the corresponding positions of the chromatogram of the test solution and the chromatogram of the control crude drug, and the negative sample has no interference. It shows that this TLC method has good specificity.

[0107] 6.2.2 TLC Identification of Cimicifugae Rhizoma

[0108] Take 0.5 g of this product, add 10 mL of methanol solution, ultrasonically treat for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution. Separately, take 1 g of the control crude drug of Cimicifugae Rhizoma, add 25 mL of distilled water, reflux and extract for 1 h, cool, filter, take the filtrate and evaporate to dryness. Dissolve the residue in 5 mL of methanol to obtain the control crude drug solution. According to the test method of thin-layer chromatography (General Rule 0502), take 6 μL of each of the above solutions, spot them on the same silica gel G plate respectively, use toluene:dichloromethane:glacial acetic acid (12:2:1) as the developing solvent, develop, take out, dry in air, spray with aluminum trichloride solution, and examine under ultraviolet light (365 nm). In the chromatogram of the test solution, fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the control crude drug.

[0109] 6.3 Methodological verification of thin-layer chromatography for Radix Astragali Praeparata Cum Melle

[0110] 6.3.1 Investigation of sample application volume

[0111] The sample treatment and development methods are the same as those under "6.2.1". Spot 6 μL, 8 μL, and 10 μL of the test solution on the same silica gel GF 254 plate respectively, and develop and examine according to the above development method.

[0112] When the sample application volume is 8 μL or more, the spots at the corresponding positions of the test solution and the control crude drug are clear and there is no other interference. Considering comprehensively, the sample application volume of 8 μL is selected.

[0113] 6.3.2 Investigation of specificity

[0114] Spot the negative sample, test solution, reference substance, and control crude drug on the same silica gel G plate respectively, develop and examine according to the above development method, and investigate the specificity of this method.

[0115] According to the development results, in the chromatogram of the test solution of Buzhong Yiqi Decoction formula granules, spots of the same color appear at the corresponding positions as those of the Radix Astragali Praeparata Cum Melle control crude drug, and there is no interference from the negative sample. It shows that the thin-layer method has good specificity.

[0116] 6.3.3 Investigation of thin-layer plates from different manufacturers

[0117] Using Qingdao Marine silica gel GF 254 plate, Qingdao Marine high-efficiency GF 254 plate, Sinopharm Group silica gel GF 254 plate, and German Merck plate as carriers respectively, develop and examine according to the above development method, and investigate the durability of different thin-layer plates.

[0118] The results show that when using different thin-layer plates (Qingdao Marine silica gel GF 254 plate, Qingdao Marine high-efficiency GF 254 plate, Sinopharm Group silica gel GF254 When developed on plates (plates from different manufacturers such as German Merck plates), the chromatograms of the test samples of the Buzhong Yiqi Decoction formula granules showed spots of the same color at the corresponding positions as those of the chromatogram of the control crude drug of Astragali Radix Praeparata Cum Melle. The thin-layer plates of each manufacturer showed clear color development. Although there were slight differences in the formation of spots, it did not affect the identification results. The resolution was good, there was no tailing phenomenon, and the background had no interference. Therefore, this method had good durability for thin-layer plates from different manufacturers.

[0119] 6.3.4 Investigation at different temperatures

[0120] The applicability of the system of this method was investigated at 5°C, 25°C, and 35°C respectively, and developed and inspected by the above-mentioned development method.

[0121] The results showed that under different temperature conditions, the test sample chromatograms showed spots of the same color at the corresponding positions as those of the control crude drug chromatograms, and the main spots were all clearly developed, the resolution was good, there was no tailing phenomenon, and the background had no interference. It indicated that temperature had no obvious influence on the thin-layer identification of Astragali Radix Praeparata Cum Melle in the Buzhong Yiqi Decoction formula granules, indicating that this thin-layer identification method had good durability to temperature.

[0122] 6.3.5 Investigation at different humidities

[0123] The applicability of the system of this method was investigated at 33%, 66%, and 88% humidity respectively, and developed and inspected by the above-mentioned development method.

[0124] The results showed that under different humidity conditions, the test sample chromatograms showed spots of the same color at the corresponding positions as those of the control crude drug chromatograms, and the main spots were all clearly developed, the resolution was good, there was no tailing phenomenon, and the background had no interference. It indicated that humidity had no obvious influence on the thin-layer identification of Astragali Radix Praeparata Cum Melle in the Buzhong Yiqi Decoction formula granules, indicating that this thin-layer identification method had good durability to humidity.

[0125] 6.4 Methodology verification of the thin-layer chromatography method for Cimicifugae Rhizoma

[0126] 6.4.1 Investigation of sample application volume

[0127] The sample treatment method was the same as that under "6.2.2". 2 μl, 4 μl, and 6 μl of the test samples were spotted on the same silica gel G plate respectively, and developed and inspected by the above-mentioned development method.

[0128] The results showed that when the sample application volume was 6 μl, the spots at the corresponding positions of the test samples and the control crude drug were clear and there was no other interference. Considering comprehensively, the sample application volume of 6 μl was selected.

[0129] 6.4.2 Investigation of specificity

[0130] Negative samples, test samples, and control crude drugs were spotted on the same silica gel G plate respectively, and developed and inspected by the above-mentioned development method to investigate the specificity of this method.

[0131] The results showed that the spots of the test sample chromatogram at the corresponding positions were the same color as those of the Cimicifugae Rhizoma control medicinal material chromatogram, and there was no interference from the negative sample. This indicated that the TLC method had good specificity.

[0132] 6.4.3 Investigation of different TLC plates

[0133] Using Qingdao Marine Silica Gel G plate, Qingdao Marine High-efficiency G plate, Sinopharm Group Silica Gel G plate and German Merck plate as carriers respectively, developing and inspecting with the above-mentioned developing method to investigate the durability of different TLC plates.

[0134] The results showed that when developed using different TLC plates (Qingdao Marine Silica Gel G plate, Qingdao Marine High-efficiency G plate, Sinopharm Group Silica Gel G plate, German Merck plate), the spots of the test sample chromatogram at the corresponding positions were the same color as those of the control medicinal material chromatogram. The spots developed clearly on the TLC plates of each manufacturer. Although there were slight differences in the spot formation, it did not affect the identification results. The resolution was good, there was no trailing phenomenon, and the background had no interference. Therefore, this method had good durability for TLC plates from different manufacturers.

[0135] 6.4.4 Investigation of temperature

[0136] The applicability of the system of this method was investigated at 5°C, 25°C, and 35°C respectively, and developed and inspected with the above-mentioned developing method.

[0137] The results showed that under different temperature conditions, the spots of the test sample chromatogram at the corresponding positions were the same color as those of the control medicinal material chromatogram, and the main spots developed clearly. The resolution was good, there was no trailing phenomenon, and the background had no interference. This indicated that temperature had no obvious effect on the TLC identification of Cimicifugae Rhizoma in the Buzhong Yiqi Decoction formula granules, showing that the TLC identification method had good durability to temperature.

[0138] 6.4.5 Investigation of humidity

[0139] The applicability of the system of this method was investigated at 33%, 66%, and 88% humidity respectively, and developed and inspected with the above-mentioned developing method.

[0140] The results showed that under different humidity conditions, the spots of the test sample chromatogram at the corresponding positions were the same color as those of the control medicinal material chromatogram, and the main spots developed clearly. The resolution was good, there was no trailing phenomenon, and the background had no interference. This indicated that humidity had no obvious effect on the TLC identification of Cimicifugae Rhizoma in the Buzhong Yiqi Decoction formula granules, showing that the TLC identification method had good durability to humidity.

[0141] 7. Determination of the content of the finished product of Buzhong Yiqi Decoction formula granules

[0142] 7.1 Determination of the preparation method of the test solution

[0143] Take the product under the filling quantity, mix well, grind finely, take 1 g, weigh accurately, place it in a 100 mL conical flask, accurately add 25 mL of 80% methanol, reflux for 40 min, cool, weigh again, make up the lost weight with 80% methanol, shake well and filter to obtain the solution.

[0144] 7.2 Determination of chromatographic conditions

[0145] Use octadecylsilane chemically bonded silica gel as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and carry out elution according to the following table; the temperature is 30 °C; the flow rate is 0.3 mL / min; the detection wavelengths are 283 nm for hesperidin and 251 nm for glycyrrhizic acid; the injection volume is 1 μL. The theoretical plate number calculated by the hesperidin peak should be not less than 2000.

[0146] Table 19 Elution gradient

[0147]

[0148] 7.3 Determination of the contents of three batches of pilot-scale finished products of Buzhong Yiqi Decoction formula granules

[0149] Weigh 1 g of samples of each batch of Buzhong Yiqi Decoction formula granules, prepare the test solution according to the method under "7.1", inject 1 μL according to the chromatographic conditions under "7.2", and calculate the content. The results are shown in the following table.

[0150] Table 20 Results of the determination of the contents of three batches of Buzhong Yiqi Decoction formula granules

[0151]

[0152] According to the results of the determination of the contents of three batches of Buzhong Yiqi Decoction formula granules, there are no significant differences in the contents of hesperidin and glycyrrhizic acid in the three batches of Buzhong Yiqi Decoction formula granules. The average value of hesperidin is 3.88 mg / g, the range of average value ± 30% is 2.72 - 5.05 mg / g, and the range of average value ± 3SD is 3.84 - 3.92 mg / g; the average value of glycyrrhizic acid is 2.75 mg / g, the range of average value ± 30% is 1.39 - 3.58 mg / g, and the range of average value ± 3SD is 2.66 - 2.84 mg / g. Since the content range of average value ± 3SD is relatively narrow and is not sufficient to be used as the basis for the quality standard limits of hesperidin and glycyrrhizic acid in Buzhong Yiqi Decoction formula granules and finished products, the average value ± 30% is used as the quality standard limits of hesperidin and glycyrrhizic acid in Buzhong Yiqi Decoction formula granules and finished products.

[0153] In summary, the beneficial effects of the present invention are as follows: The granule prepared by the present invention has rapid drug absorption, high bioavailability, good stability, is convenient for taking, carrying and storing, has accurate and easy-to-control dosage, and has a simple production process and low cost. Functional indications: Tonifying the middle-jiao and replenishing qi, lifting yang and raising prolapse, used for diarrhea, rectal prolapse, uterine prolapse caused by weakness of the spleen and stomach and sinking of middle qi, manifested as lassitude, anorexia, abdominal distension, loose stools and chronic diarrhea, anal prolapse or uterine prolapse.

[0154] There are mainly three common granulation methods for traditional Chinese medicine granules, including dry granulation, wet granulation and fluidized bed granulation. The dry granulation method has high requirements for production instruments and equipment, long production time, large energy consumption, so the production cost is relatively high; the granules prepared by wet granulation are easy to adhere into blocks during the drying process, affecting the granule yield, and the prepared granules have a small bulk density; fluidized bed granulation can complete the processes of mixing, granulation, drying, etc. in one step under a closed state, simplify the process flow, avoid dust flying, and has good fluidity. At the same time, it is especially suitable for heat-sensitive materials, and the process parameters are stable and highly repeatable, suitable for continuous batch production.

[0155] The present invention finally adopts the fluidized bed granulation method to prepare granules. After verification, the formability is good, and the granule preparation process is stable and feasible, which can lay a solid foundation for the subsequent research and development of enterprises. The thin-layer identification method adopted has good durability and accurate identification. And it is determined that the content of dried tangerine peel calculated as hesperidin (C 28 H 34 O 15 ) in this product should not be less than 2.72 mg / g, and the content of roasted licorice root calculated as glycyrrhizic acid (C 42 H 62 O 16 ) should not be less than 1.93 mg / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Figure 1 It is the thin-layer identification result of Buzhong Yiqi Decoction granules; A is the thin-layer identification result of Cimicifuga foetida; among which 1: negative test sample (lacking Cimicifuga foetida) 2: control medicinal material of Cimicifuga foetida 3: test sample of Buzhong Yiqi Decoction formula granules; B is the thin-layer identification result of Astragalus membranaceus; among which 1: negative test sample (lacking Astragalus membranaceus) 2: control medicinal material of Astragalus membranaceus 3: test sample of Buzhong Yiqi Decoction formula granules. DETAILED DESCRIPTION OF THE INVENTION

[0157] The following further details the specific embodiments of the present invention, but the present invention is not limited to these embodiments. Any improvement or substitution based on the basic spirit of this embodiment still falls within the scope protected by the claims of the present invention.

[0158] Example 1

[0159] 1. A Buzhong Yiqi Decoction granule, and the prescription of the Buzhong Yiqi Decoction granule is as follows: roasted Astragalus membranaceus 659 g, Codonopsis pilosula 198 g, roasted Glycyrrhiza uralensis 329 g, stir-fried Atractylodes macrocephala Koidz. with bran 198 g, Angelica sinensis 198 g, Rhizoma Cimicifugae 198 g, Bupleurum chinense 198 g, Pericarpium Citri Reticulatae 198 g, Zingiber officinale 66 g, and Ziziphus jujuba 132 g.

[0160] 2. The preparation method of the above Buzhong Yiqi Decoction granule, comprising the following steps:

[0161] (1) Soak the 10 herbs in water for 0.5 h and then decoct them three times, each decoction for 2 h, and combine the three decoction liquids; the water addition amount for the first decoction is 10 times the mass of all the herbs, and the water addition amounts for the second and third decoctions are both 8 times the mass of all the herbs.

[0162] (2) Filter the combined decoction liquid, concentrate the filtrate into a clear paste, and then mix it with 450 - 570 g of auxiliary materials to prepare the granule by the boiling granulation method.

[0163] Further, the relative density of the clear paste is 1.10 - 1.15.

[0164] Further, the auxiliary materials are composed of maltodextrin and soluble starch in a mass ratio of 1:3.

[0165] Further, the content of hesperidin in the granule is not less than 2.72 mg / g, and the content of glycyrrhizic acid is not less than 1.93 mg / g.

[0166] Perform identification and determination on the granule obtained in Example 1

[0167] 1. Identification

[0168] (1) Take 0.5 g of this product, add 10 mL of methanol solution, ultrasonically treat for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution. Additionally, take 1 g of the control crude drug of Rhizoma Cimicifugae, add 25 mL of distilled water, reflux and extract for 1 h, cool, filter, take the filtrate and evaporate to dryness. Dissolve the residue in 5 mL of methanol to obtain the control crude drug solution. According to the thin-layer chromatography method (General Rule 0502) test, suck 6 μL of each of the above solutions and spot them on the same silica gel G plate respectively, use toluene:dichloromethane:glacial acetic acid (12:2:1) as the developing agent, develop, take out, dry, spray with aluminum trichloride solution, and view under an ultraviolet lamp (365 nm). In the test solution chromatogram, at the corresponding position to the control crude drug chromatogram, there are fluorescent spots showing the same color, and the results are as Figure 1 shown in -A.

[0169] (2) Take 0.5 g of this product, add 10 mL of methanol solution, ultrasonically treat for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution. Separately, take 1 g of the control crude drug of stir-fried Astragalus membranaceus, add 25 mL of distilled water, reflux and extract for 1 h, cool, filter, take the filtrate and evaporate to dryness. Dissolve the residue in 5 mL of methanol to obtain the control crude drug solution. According to the test method of thin-layer chromatography (General Principles 0502), accurately pipette 8 μL of each of the above solutions, and spot them respectively on the same GF 254 silica gel plate. Use chloroform-methanol (10:1) as the developing solvent, develop, take out, air dry, and examine under ultraviolet light (254 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the control crude drug, fluorescent spots of the same color shall appear. The results are as shown in Figure 1 -B.

[0170] 2. Content determination: Determine according to the high performance liquid chromatography method (General Principles 0512)

[0171] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the detection wavelength for hesperidin is 283 nm and for glycyrrhizic acid is 251 nm. The number of theoretical plates calculated based on the hesperidin peak shall not be less than 2000.

[0172]

[0173] 2.1 Preparation of reference solution: Take an appropriate amount of hesperidin reference substance, accurately weigh it, dissolve it in methanol to prepare a reference solution containing 0.40 mg per 1 mL, and that's it. Separately, take an appropriate amount of ammonium glycyrrhizinate reference substance, accurately weigh it, dissolve it in 70% ethanol to prepare a reference solution containing 0.2 mg of ammonium glycyrrhizinate per 1 mL, and that's it (weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207).

[0174] 2.2 Preparation of test solution: Take this product under the filling quantity item, mix well, grind it finely, take 1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, ultrasonically extract for 40 min, cool, weigh again, make up the lost weight with 80% methanol, shake well and filter to obtain the test solution.

[0175] 2.3 Determination method: Accurately pipette 1 μL of each of the reference solution and the test solution respectively, inject them into the liquid chromatograph for determination, and that's it.

[0176] This product contains tangerine peel calculated as hesperidin (C 28 H 34 O 15 ) shall not be less than 2.72 mg / g, and contains stir-fried licorice root calculated as glycyrrhizic acid (C 42 H 62 O 16)It should be not less than 1.93 mg / g.

[0177] 3. Investigation on the Stability of Pilot-scale Production Samples of Granules

[0178] 3.1 Method

[0179] Refer to the General Principles for the Stability Testing of Drug Substances and Preparations 9001 in the Chinese Pharmacopoeia (2020 Edition) to conduct stability tests on the Buzhong Yiqi Decoction formula granules, including stress stability tests, accelerated stability tests, and long-term stability tests.

[0180] 3.2 Stress Tests

[0181] 3.2.1 High Temperature Test

[0182] Take the pilot-scale samples of 211101, pack them in the commercially available aluminum foil composite film, and place them in a constant temperature equipment at 60°C ± 2°C. Sampling is carried out on the 0th day, 5th day, 10th day, and 30th day respectively, and tested according to the key items for stability inspection. Compare the test results with the formulated quality standards to determine whether they meet the formulated quality standards.

[0183] 3.2.2 High Humidity Test

[0184] Take the pilot-scale samples of 211101, pack them in the commercially available aluminum foil composite film, and place them in a constant humidity equipment at 25°C and relative humidity of 90% ± 5%. Sampling is carried out on the 0th day, 5th day, and 10th day respectively, and tested according to the key items for stability inspection. Compare the test results with the formulated quality standards to determine whether they meet the formulated quality standards.

[0185] 3.2.3 Strong Light Irradiation Test

[0186] Take the pilot-scale samples of 221101, pack them in the commercially available aluminum foil composite film, and place them in a container with an illuminance of 4500 lx ± 500 lx, and the total illuminance of the light source should be not less than 1.2×10 6 lux·hr, and the energy of the near-ultraviolet lamp should be not less than 200 W·hr / m 2 . Sampling is carried out on the 0th day, 5th day, 10th day, and 30th day respectively, and tested according to the key items for stability inspection. Compare the test results with the formulated quality standards to determine whether they meet the formulated quality standards.

[0187] 3.3 Accelerated Stability Test

[0188] Take 3 batches of pilot-scale production samples, pack them in the commercially available aluminum foil composite film, and place them at a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5% for 6 months. Sampling is carried out once at the end of the 0th month, 1st month, 2nd month, 3rd month, and 6th month during the test period, and tested according to the specified stability inspection items. Compare the test results with the formulated quality standards to determine whether they meet the formulated quality standards.

[0189] 3.4 Long-term Stability Test

[0190] Take 3 batches of pilot-scale production samples and place them under the conditions of temperature 30°C ± 2°C and relative humidity 65% ± 5% for 12 months. Sampling is carried out once every 3 months, and sampling is carried out at 0 month, 3 months, 6 months, 9 months, and 12 months respectively, and tested according to the key stability inspection items. After 12 months, continuous inspection is still required. Sampling is carried out at 18 months, 24 months, and 36 months respectively for inspection, and the inspection results are compared with those at 0 month to determine the shelf life of the drug.

[0191] 3.5 Inspection Items

[0192] Key stability inspection items for Buzhong Yiqi Decoction formula granules: appearance, particle size, moisture, solubility, identification, microbial limit, content determination, etc.

[0193] 3.6 Stability Test Plan

[0194] Table 21 Stability Test Samples, Storage Conditions, and Determination Methods

[0195]

[0196] 3.7 Stability Test Results

[0197] 3.7.1 Stress Test

[0198] 3.7.1.1 High Temperature Test

[0199] Table 22 High Temperature Test Results

[0200]

[0201]

[0202] 3.7.1.2 High Humidity Test

[0203] Table 23 High Humidity Test Results

[0204]

[0205] 3.7.1.3 Strong Light Irradiation Test

[0206] Table 24 Strong Light Irradiation Test Results

[0207]

[0208]

[0209] 3.7.2 Accelerated Stability Test Results

[0210] Table 25 Results of the accelerated stability test of Buzhong Yiqi Decoction formula granules Sample packaging conditions: Medicated composite film 、Storage conditions: 40℃±2℃、 Humidity 75％±5％

[0211]

[0212]

[0213]

[0214] 3.7.3 Results of the long-term stability test

[0215] Table 26 Results of the long-term stability test of Buzhong Yiqi Decoction formula granules Sample packaging conditions: Medicated composite film 、Storage conditions: Temperature: 30°C ± 2°C, Relative humidity: 65% ± 5%

[0216]

[0217]

[0218]

[0219] From the above experimental results, it can be seen that the drug substance basis of the granules prepared by the present invention remains unchanged, and the original curative effect can be guaranteed. In addition, the granules have fast drug absorption, high bioavailability, good stability, are convenient for taking, carrying and storing, have accurate and easy-to-control dosage, and have a simple production process and low cost.

Claims

1. A Buzhong Yiqi Decoction granule, characterized in that, the prescription of the Buzhong Yiqi Decoction granule is as follows: roasted Astragalus membranaceus 650 - 670 g, Codonopsis pilosula 185 - 213 g, roasted Glycyrrhiza uralensis 310 - 337 g, stir-fried Atractylodes macrocephala with bran 185 - 213 g, Angelica sinensis 185 - 213 g, Cimicifuga foetida 185 - 213 g, Bupleurum chinense 185 - 213 g, Pericarpium Citri Reticulatae 185 - 213 g, Zingiber officinale 60 - 72 g, Ziziphus jujuba 130 - 135 g; The preparation method of the Buzhong Yiqi Decoction granule described above includes the following steps: (1) Soak the 10 herbs in water for 0.5 h and then decoct them three times, each decoction for 2 h, and combine the three decoction liquids; The water addition amount for the first decoction is 10 times the mass of all the herbs, and the water addition amounts for the second and third decoctions are both 8 times the mass of all the herbs; (2) Filter the combined decoction liquid, concentrate the filtrate into a clear paste, the relative density of the clear paste is 1.10 - 1.15, and then mix it with 450 - 570 g of auxiliary materials, and prepare the granule by the boiling granulation method; The auxiliary materials are composed of maltodextrin and soluble starch in a mass ratio of 1:

3.

2. The identification method of a Buzhong Yiqi Decoction granule as described in claim 1, characterized in that, it includes the following steps: (1) Take 0.5 g of the granule, add 10 mL of methanol solution, ultrasonically treat for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution; take another 1 g of the control medicinal material of Cimicifuga foetida, add 25 mL of distilled water, reflux and extract for 1 h, cool, filter, take the filtrate and evaporate to dryness; dissolve the residue in 5 mL of methanol to obtain the control medicinal material solution; according to the thin-layer chromatography test, absorb 6 μL of each of the above solutions, respectively spot them on the same silica gel G plate, develop, take out, dry in the air, spray with aluminum trichloride solution, and examine under an ultraviolet lamp; (2) Take 0.5 g of the granule, add 10 mL of methanol solution, ultrasonically treat for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution; additionally, take 1 g of the control crude drug of stir-fried Astragali Radix, add 25 mL of distilled water, reflux and extract for 1 h, cool, filter, take the filtrate and evaporate to dryness; dissolve the residue in 5 mL of methanol to obtain the control crude drug solution; according to the thin-layer chromatography test, absorb 8 μL of each of the above solutions, respectively spot them on the same GF 254 silica gel plate, develop, take out, air dry, and examine under an ultraviolet lamp.

3. The identification method of a Buzhong Yiqi Decoction granule as described in claim 2, characterized in that, in the step (1), toluene, dichloromethane and glacial acetic acid are used as the developing agent, toluene:dichloromethane:glacial acetic acid = 12:2:1; in step 2, chloroform and methanol are used as the developing agent, chloroform:methanol = 10:

1.

4. The content determination method of a Buzhong Yiqi Decoction granule as described in claim 1, characterized in that, it is determined by high performance liquid chromatography, and the specific steps are as follows: (1) Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the detection wavelength for hesperidin is 283 nm and for glycyrrhizic acid is 251 nm; the theoretical plate number calculated based on the hesperidin peak should be not less than 2000; (2) Prepare the reference substance solution: Take an appropriate amount of hesperidin reference substance, accurately weigh it, and make a reference substance solution containing 0.40 mg per 1 mL with methanol, that is obtained; take another appropriate amount of ammonium glycyrrhizinate reference substance, accurately weigh it, add 70% ethanol to make a reference substance solution containing 0.2 mg of ammonium glycyrrhizinate per 1 mL, that is obtained; (3) Preparation of the test solution: Take the product under the filling quantity, mix well, grind finely, take 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, extract ultrasonically for 40 min, cool, weigh again, make up the lost weight with 80% methanol, shake well and filter to obtain the solution; (4) Determination: Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination to obtain the results.

5. The method for determining the content of the Buzhong Yiqi Decoction granule as described in claim 4, characterized in that the content of hesperidin in the granule is not less than 2.72 mg / g, and the content of glycyrrhizic acid is not less than 1.93 mg / g.

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