Preparation method and quality control method of fingered citron formula granules

Abstract

The embodiment of the invention discloses a preparation method and a quality control method of fingered citron formula granules. The preparation method of the fingered citron formula granules comprises the following steps: crushing fingered citron decoction pieces, carrying out alcohol extraction twice, filtering, combining filtrates, carrying out vacuum concentration until no alcohol smell exists, and adding water to obtain a concentrated solution; enabling the concentrated solution to flow through a macroporous adsorption resin column for adsorption and alcohol elution, and collecting eluent; and concentrating the eluent under reduced pressure to obtain an extract, adding auxiliary materials, uniformly mixing, granulating, drying, and finishing to obtain the fingered citron formula granules. The preparation method provided by the invention can enrich hesperidin and other active ingredients, the prepared fingered citron formula granules are high in active ingredient content, and the preparation method is simple, high in operability and suitable for expanded production. The quality control method provided by the invention is accurate, reliable and good in repeatability, and can comprehensively evaluate the quality of the fingered citron formula granules.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of preparation of traditional Chinese medicine preparations, and specifically to a preparation method of bergamot formula granules and a quality control method thereof. Background Art

[0002] Buddha's hand is the dried fruit of the Rutaceae plant Buddha's hand. Mature Buddha's hand has the effects of drying dampness and resolving phlegm, soothing the liver and regulating qi, and harmonizing the stomach and relieving pain. Buddha's hand mainly contains flavonoids, coumarins, limonoids, volatile oils and polysaccharides.

[0003] In the "National Drug Standards for Chinese Medicine Formula Granules (First Batch)", the bergamot formula granules are extracted by boiling bergamot with water, and the extract is concentrated, granulated, and dried to make 1g of formula granules equivalent to 1.6g of bergamot slices. Its quality control is based on the content of hesperidin. Bergamot contains a variety of chemical components, and it is difficult to reflect its quality by measuring the content of a single component. Only by further measuring the content of multiple indicator components can the efficacy of bergamot formula granules and related preparations be guaranteed. In addition, its thin layer identification uses bergamot reference medicinal materials for identification, and this method lacks comparison with reference products.

[0004] CN 111912916A is the research result of the applicant before, which discloses a method for determining the content of index components in a bergamot preparation, which can simultaneously detect three index components of diosmin, hesperidin, and 5,7-dimethoxycoumarin in bergamot preparations, and has the advantages of accurate detection results, high precision, and good repeatability. The prior art shows that limonin and nomilin have multiple biological activities such as anti-tumor, anti-oxidation, anti-inflammatory, anti-neurogenic disease, immunomodulation, insecticide, antibacterial, and antiviral, and have good application prospects. Therefore, the detection of limonin and nomilin index components is also of great significance for improving the quality of bergamot preparation products. However, the study found that when the high-performance liquid chromatography detection conditions in the method are used to simultaneously detect diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin, the peak time of limonin and nomilin is close to that of 5,7-dimethoxycoumarin, and it is difficult to accurately determine the content of each component, and there are problems such as long separation time and large baseline drift.

[0005] In view of this, the present invention application is specially proposed. Summary of the invention

[0006] To this end, an embodiment of the present invention provides a method for preparing bergamot formula granules and a method for controlling their quality.

[0007] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0008] According to a first aspect of an embodiment of the present invention, the present invention provides a method for preparing bergamot formula particles, comprising the following steps:

[0009] (1) crushing the bergamot slices, extracting with alcohol twice, filtering, combining the filtrates, concentrating under reduced pressure until there is no alcohol taste, adding water, and obtaining a concentrated solution;

[0010] (2) passing the concentrated solution through a macroporous adsorption resin column for adsorption, eluting with alcohol, and collecting the eluate;

[0011] (3) The eluate is concentrated under reduced pressure to an extract, auxiliary materials are added and mixed evenly, and the extract is granulated, dried, and sized to obtain the bergamot formula granules.

[0012] Furthermore, in step (1), the alcohol extraction conditions are: reflux extraction twice with 70% ethanol in an amount 8-12 times the volume of the bergamot slices, each time for 1-2 hours; and adding water to a crude drug concentration of 0.4-0.6 g / ml.

[0013] Furthermore, in step (2), the model of the macroporous adsorption resin column is AB-8, the mass ratio of the bergamot slices to the resin is 1:1.8-4, the column is wet packed with water, the diameter-to-height ratio is 1:3-5, and the adsorption time is 15-30 minutes;

[0014] The alcohol elution condition is: washing with 7-10 column volumes of water until the effluent has no Molish reaction, and then eluting with 5-6 column volumes of 70% ethanol.

[0015] Alcohol elution includes washing away sugar and other impurities with water, and then desorbing and enriching the effective ingredients. The inventor compared the cough and expectorant and gastrointestinal motility-promoting effects of different elution parts of bergamot, and found that the cough and expectorant effect of 70% ethanol eluate was better, while the gastrointestinal motility-promoting effect of 50% ethanol eluate was better. The compounds in different elution parts of bergamot were further identified, and a total of 42 components were identified, including 37 components in the 70% ethanol extract, 17 components in the water eluate, 36 components in the 50% ethanol eluate, and 42 components in the 70% ethanol eluate, indicating that the chemical components are mainly in the 70% ethanol eluate, so the present invention uses 70% ethanol elution.

[0016] Furthermore, in step (3), 0.2-0.3 times the mass of dextrin of the bergamot slices is added and mixed evenly, 0.1-0.2 times the mass of bergamot slices of 70% ethanol is used as a wetting agent for wet granulation, granulation is carried out using a No. 1 sieve, and drying is carried out at 50-60° C. for 60-90 minutes. Granules that can pass through the No. 1 sieve and cannot pass through the No. 5 sieve are defined as qualified granules, thereby obtaining bergamot formula granules.

[0017] According to a second aspect of an embodiment of the present invention, the present invention provides a bergamot formula granule, which is made by the method described in any one of the above items.

[0018] According to a third aspect of an embodiment of the present invention, the present invention provides a quality control method for the bergamot formula granules as described above, comprising:

[0019] A step of performing thin layer chromatography to identify hesperidin and 5,7-dimethoxycoumarin in the bergamot formula granules; and

[0020] The invention relates to a step of determining the contents of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin and nomilin in the bergamot formula granules by high performance liquid chromatography.

[0021] Furthermore, the step of thin layer chromatography identification comprises:

[0022] (1) Preparation of test solution: 0.2 g of the bergamot formula granules were added with 20 ml of methanol, ultrasonicated, cooled, filtered, the filtrate was evaporated to dryness, and 1 ml of methanol was added to the residue as the test solution;

[0023] (2) Preparation of reference solution: Take hesperidin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the hesperidin reference substance solution; take 5,7-dimethoxycoumarin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the 5,7-dimethoxycoumarin reference substance solution;

[0024] (3) Determination:

[0025] 2-10 μl of the test solution and hesperidin reference solution were taken and spotted on the same silica gel plate, respectively, and the lower layer solution of chloroform-ethyl acetate-methanol-water in a volume ratio of 3:8:4:2 was used as the developing solvent. The plate was taken out, dried, sprayed with 1% aluminum chloride ethanol solution, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference substance.

[0026] Pipette 2-10 μl of the test solution and 5,7-dimethoxycoumarin reference solution, respectively, and spot them on the same silica gel plate. Use n-hexane-acetone-chloroform in a volume ratio of 10:6:1 as the developing solvent. Develop the plate, take out, dry it, and examine it under a 365nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference substance.

[0027] Furthermore, in step (3), the spotting volume of the test solution and the hesperidin reference solution is 2 μl;

[0028] The silica gel plate is a hand-laid plate or a silica gel G plate, and the unfolding environment is: temperature 10-26° C., relative humidity 32.0%-72.0%.

[0029] Furthermore, the step of high performance liquid chromatography content determination comprises:

[0030] (1) Preparation of mixed reference substance solution: accurately weigh appropriate amounts of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin reference substances, dissolve them in methanol, make up to volume, and shake well to obtain reference substance stock solutions with concentrations of 857.5, 655.2, 698.7, 816.6, and 502.0 μg / ml, respectively;

[0031] Take 1 ml of each reference substance stock solution and place it in a 10 ml volumetric flask, add methanol to make up to volume to obtain a mixed standard solution;

[0032] The mixed standard solution was diluted 2, 4, 8, and 10 times by the multiple dilution method to prepare mixed reference solutions of different concentrations;

[0033] (2) Preparation of the test solution: accurately weigh 0.2 g of the bergamot formula granules, place in a stoppered conical flask, add 20 ml of methanol, weigh, ultrasonicate, cool, add methanol to make up the weight loss, filter, and take the filtrate to obtain the test solution;

[0034] (3) Determination: 2-10 μl of the test solution and the mixed reference solution were respectively taken and injected into a liquid chromatograph for determination. The chromatographic conditions were: the chromatographic column was C 18 The filler was octadecylsilane bonded silica gel, with 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution: 0-8min, 20% B; 8-10min, 20%-30% B; 10-15min, 30% B; 15-17min, 30%-53% B, 17-25min, 53% B, the flow rate was 1ml / min, the column temperature was 30℃, and the liquid chromatograph detectors were DAD and CAD.

[0035] Furthermore, in step (3), the detection wavelength of the DAD detector is 254 nm; the atomization temperature of the CAD detector is 35° C., the acquisition frequency is 10 Hz, and the filtration constant is 5.0 s.

[0036] The embodiments of the present invention have the following advantages:

[0037] (1) The preparation method provided by the present invention can enrich effective ingredients such as hesperidin, and the prepared bergamot formula granules have a high content of effective ingredients. The preparation method is simple, highly operable, and suitable for large-scale production.

[0038] (2) The quality control method provided by the present invention includes a thin layer chromatography identification method and a high performance liquid chromatography content determination method, wherein the thin layer chromatography method uses 5,7-dimethoxycoumarin and hesperidin as reference substances for identification, respectively; when the high performance liquid chromatography method simultaneously determines the five index components of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin in the bergamot formula granules, limonin and nomilin have very weak ultraviolet absorption and are difficult to detect using a conventional DAD detector. The present invention uses a DAD in series with a CAD detector and optimizes the elution conditions at the same time, so as to simultaneously determine the contents of the above five index components, has the advantages of accuracy, reliability, and good repeatability, and can comprehensively evaluate the quality of the bergamot formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0040] Figure 1 A schematic diagram of the results of investigating different developing agents in thin layer chromatography identification using hesperidin as a reference substance provided by the present invention;

[0041] Figure 2 A schematic diagram of the results of investigating different developing agents in thin layer chromatography identification using 5,7-dimethoxycoumarin as a reference substance provided by the present invention;

[0042] Figure 3 A schematic diagram of the results of different sample amounts in thin layer chromatography identification using hesperidin as a reference substance provided by the present invention;

[0043] Figure 4 A schematic diagram of the results of different sample amounts in thin layer chromatography identification using 5,7-dimethoxycoumarin as a reference substance provided by the present invention;

[0044] Figure 5 A schematic diagram of the results of the durability test in the thin layer chromatography identification using hesperidin as a reference substance provided by the present invention;

[0045] Figure 6 A schematic diagram of the results of the durability test in thin layer chromatography identification using 5,7-dimethoxycoumarin as a reference substance provided by the present invention;

[0046] Figure 7 A schematic diagram of the thin layer chromatography identification results of hesperidin in the bergamot formula granules (3 batches) provided by the present invention;

[0047] Figure 8A schematic diagram of the thin layer chromatography identification results of 5,7-dimethoxycoumarin in the Buddha's Hand formula granules (3 batches) provided by the present invention;

[0048] Fig. 9 The present invention provides an overlay of the Buddha's hand fingerprint spectrum and a reference fingerprint spectrum, wherein peak number 11: diosmin; peak number 12: hesperidin; peak number 17: 5,7-dimethoxycoumarin;

[0049] Fig.10 The HPLC diagram of the mixed reference substance and test substance solution provided by the present invention, wherein peak number 1: diosmin; peak number 2: hesperidin; peak number 3: 5,7-dimethoxycoumarin; peak number 4: limonin; peak number 5: nomiline, A: reference substance (DAD detector); B: reference substance (CAD detector); C: test substance (DAD detector); D: test substance (CAD detector);

[0050] Fig.11 HPLC diagram for screening of four mobile phase conditions for the Buddha's hand formula granules provided by the present invention; A: DAD detector; B: CAD detector. DETAILED DESCRIPTION

[0051] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Example 1

[0053] Optimization of orthogonal reflux extraction conditions

[0054] The ethanol reflux extraction method was used. The total extraction amount of representative flavonoid components of bergamot (diosmin and hesperidin), representative coumarin components (5,7-dimethoxycoumarin) and representative limonoid components (limonin and nomilin) ​​were selected as reference indicators. The volume fraction of ethanol, solid-liquid ratio and reflux extraction time were used as the investigation factors. According to L9(3 4) Orthogonal test table for reflux extraction experiment, factor level table is shown in Table 1, and orthogonal test results are shown in Table 2. The range R in the orthogonal test result table can reflect the influence of each factor on the index. The larger the R value, the greater the influence on the test results. The range results are shown in Table 2, RA>RC>RB, that is, the factors affecting the extraction effect of bergamot are ranked in the order of ethanol concentration, extraction time, and material-liquid ratio. The results of variance analysis are shown in Table 3. The order of influence of each factor on the total extraction rate within the investigation interval is C>A>B, and the differences between different levels of each factor are not statistically significant (P>0.05).

[0055] The extraction conditions are determined as follows: A2B2C2, i.e., adding 20 times the amount of 70% ethanol, reflux extraction for 1.5 hours, and extracting twice each time. However, the influence factor of the solid-liquid ratio is relatively low, so the solid-liquid ratio is 1:10, which reduces the use of extraction solvents and thus reduces production costs.

[0056] Table 1 Reflux extraction factor level table

[0057]

[0058] Table 2 Orthogonal test results

[0059]

[0060] Table 3 Orthogonal test variance analysis results

[0061]

[0062] Example 2

[0063] This embodiment provides a method for preparing bergamot formula granules, comprising the following steps:

[0064] (1) Add 10 times 70% ethanol to the citron slices for reflux extraction twice, each reflux for 1.5 hours, filter after the reflux is completed, combine the filtrates, recover the ethanol from the filtrates and concentrate under reduced pressure until there is no alcohol taste, add water to the crude drug concentration of 0.5 g / ml, and obtain a concentrated solution for standby use;

[0065] (2) passing the concentrated solution obtained in step (1) through an AB-8 macroporous adsorption resin column, wherein the mass ratio of the resin to the bergamot slices is 2:1, the column is wet packed, the diameter-to-height ratio is 1:4, the adsorption time is 15 minutes, and after loading, eluting with 7 column volumes of water until there is no Molish reaction in the effluent, and then eluting with 5 column volumes of 70% ethanol, collecting the eluate for standby use;

[0066] (3) The eluate obtained in step (2) is concentrated under reduced pressure to an extract, 0.2 times the mass of dextrin of the citron slice is added and mixed evenly, 0.1 times the mass of 70% ethanol of the citron slice is used as a wetting agent for wet granulation (granulation is performed using a No. 1 sieve), dried at 50° C. for 90 minutes, and granulated (it is specified that granules that can pass through a No. 1 sieve and cannot pass through a No. 5 sieve are qualified granules), thereby obtaining citron formula granules.

[0067] Example 3

[0068] Thin layer chromatography identification experiment of bergamot formula granules

[0069] 1. Reference substances and samples

[0070] Table 4 Reference substance and sample information

[0071]

[0072] 2. Methods and Results

[0073] 2.1 Preparation of reference solution:

[0074] Take the hesperidin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the hesperidin reference substance solution; take the 5,7-dimethoxycoumarin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the 5,7-dimethoxycoumarin reference substance solution.

[0075] 2.2 Preparation of test solution:

[0076] Take 0.2 g of bergamot formula granules (prepared according to the method of Example 2), add 20 ml of methanol, treat with ultrasound (frequency 40 KHz) for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 1 ml of methanol to the residue, dissolve it, and use it as the test solution.

[0077] 2.3 Screening of developing agents

[0078] 2.3.1 Optimization of hesperidin developing agent conditions

[0079] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 2 μl of the test solution and hesperidin reference solution were taken and spotted on the same silica gel G thin layer plate in turn, marked as A, B, and C, and the lower layer solution placed at 4°C was developed with different developing systems, namely, developing agent A: ethyl acetate-methanol-water (volume ratio of 100:17:13), developing agent B: ethyl acetate-formic acid-water (volume ratio of 10:1:1), and developing agent C: chloroform-ethyl acetate-methanol-water (volume ratio of 3:8:4:2). The plate was taken out, dried, sprayed with 1% aluminum chloride ethanol solution, dried, and inspected under ultraviolet light (365 nm).

[0080] Results Figure 1In the chromatogram of the test sample, in the developing agent A, the test sample shows a fluorescent spot of the same color at the corresponding position of the chromatogram of the reference sample, but the color is lighter and the spot is not clear; in the developing agent B, the test sample has no fluorescent spot of the same color at the corresponding position of the chromatogram of the reference sample; in the developing agent C, the test sample shows a fluorescent spot of the same color at the corresponding position of the chromatogram of the reference sample. Therefore, the lower layer solution of the developing agent C: chloroform-ethyl acetate-methanol-water (volume ratio 3:8:4:2) placed at 4℃ is the best.

[0081] 2.3.2 Optimization of Developing Conditions of 5,7-Dimethoxycoumarin

[0082] According to the thin layer chromatography method (General Rule 0502 of the 2020 edition of the Chinese Pharmacopoeia), 2 μl of the test solution and 5,7-dimethoxycoumarin reference solution were taken and spotted on the same silica gel G thin layer plate in turn, marked as Ⅰ, Ⅱ, and Ⅲ, and developed with different developing systems, namely, developing agent Ⅰ: n-hexane-acetone-chloroform (volume ratio of 10:6:1), developing agent Ⅱ: cyclohexane-ethyl acetate-formic acid (volume ratio of 9:6:1), and developing agent Ⅲ: n-hexane-ethyl acetate-formic acid (volume ratio of 9:6:1). Take out, dry, and inspect under ultraviolet lamp (365 nm).

[0083] Results Figure 2 In the chromatogram of the test sample, in the developing agent I, 5 spots can be observed in the chromatogram of the test sample, and the test sample shows fluorescent spots of the same color at the corresponding position of the chromatogram of the reference sample, and the other spots are well distributed; in the developing agent II, 4 spots can be observed in the chromatogram of the test sample, and the test sample spots show fluorescent spots of the same color at the corresponding positions of the reference sample, but the other spots are tailing; in the developing agent III, 3 spots can be observed in the chromatogram of the test sample, and the test sample spots show fluorescent spots of the same color at the corresponding positions of the reference sample, but the other spots are tailing. Compared with developing agents II and III, developing agent I can observe more spots without tailing. Overall, developing agent I: n-hexane-acetone-chloroform (volume ratio 10:6:1) is the best.

[0084] 2.4 Sampling quantity inspection

[0085] 2.4.1 Hesperidin

[0086] Respectively take 1 μl, 2 μl and 5 μl of the test solution and reference solution, and use the lower layer solution of the best developing system chloroform-ethyl acetate-methanol-water (volume ratio 3:8:4:2) at 4°C as the developing agent. Develop, take out, dry, spray 1% aluminum chloride ethanol solution, dry, and inspect under ultraviolet light (365nm).

[0087] Results Figure 3In the figure, serial numbers 1-3 are hesperidin reference substances, and the spotting amounts are 1μl, 2μl, and 5μl respectively. Serial numbers 4-6 are test solutions, and the spotting amounts are 1μl, 2μl, and 5μl respectively. In the figure, the best thin layer effect is achieved when the spotting amount is 2μl.

[0088] 2.4.25,7-Dimethoxycoumarin

[0089] 1 μl, 2 μl and 5 μl of the test solution and reference solution were respectively taken, and the optimal developing system of n-hexane-ethyl acetate-formic acid (volume ratio 9:6:1) was used as the developing agent. The tube was developed, taken out, dried and examined under ultraviolet light (365 nm).

[0090] Results Figure 4 In the figure, serial numbers 1-3 are 5,7-dimethoxycoumarin reference substances, and the spotting amounts are 1μl, 2μl, and 5μl, respectively. 4-6 are test solutions, and the spotting amounts are 1μl, 2μl, and 5μl, respectively. In the figure, the best thin layer effect is achieved when the spotting amount is 2μL.

[0091] 2.5 Durability inspection of the development environment

[0092] The effects of different silica gel plates (hand-laid plates, silica gel G plates), different developing temperatures (26°C, 10°C) and different relative humidity (32.0%, 72.0%) on the behavior of thin layer chromatography were investigated.

[0093] 2.5.1 Study on the durability of hesperidin

[0094] Separately take 2 μl of the test solution and reference solution, and use the lower layer solution of the best developing system chloroform-ethyl acetate-methanol-water (volume ratio 3:8:4:2) at 4°C as the developing agent. Develop, take out, dry, spray with 1% aluminum chloride ethanol solution, dry, and examine under ultraviolet light (365 nm).

[0095] Results Figure 5 The results showed that different silica gel plates had no significant effect on the thin layer chromatography identification method; the temperature was in the range of 10 to 26°C and the relative humidity was in the range of 32.0% to 72.0%, which had no significant effect on the thin layer identification.

[0096] 2.5.25,7-Dimethoxycoumarin Durability Study

[0097] 2 μl of the test solution and reference solution were taken respectively, and the optimal developing system of n-hexane-ethyl acetate-formic acid (volume ratio 9:6:1) was used as the developing agent. The samples were developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0098] Results Figure 6The results showed that different silica gel plates had no significant effect on the thin layer chromatography identification method; the temperature was in the range of 10 to 26°C and the relative humidity was in the range of 32.0% to 72.0%, which had no significant effect on the thin layer identification.

[0099] 2.63 batches of bergamot formula granules thin layer results

[0100] 2 μl of the test solution and hesperidin reference solution were taken respectively, and spotted on the same silica gel G thin layer plate in turn, and the lower layer solution of the best developing system chloroform-ethyl acetate-methanol-water (volume ratio of 3:8:4:2) placed at 4°C was used as the developing agent, and the plate was developed, taken out, dried, sprayed with 1% aluminum chloride ethanol solution, dried, and examined under ultraviolet lamp (365nm); 2 μl of the test solution and 5,7-dimethoxycoumarin reference solution were taken respectively, and the best developing system n-hexane-ethyl acetate-formic acid (volume ratio of 9:6:1) was used as the developing agent, and the plate was developed, taken out, dried, and examined under ultraviolet lamp (365nm).

[0101] Results Figure 7 and Figure 8 In the chromatograms of the bergamot formula granules prepared from three different batches of bergamot slices according to the method of Example 2, the same spots appeared at the corresponding positions of the chromatograms of the hesperidin and 5,7-dimethoxycoumarin reference substances, respectively. Figure 7 Serial number 1 is the hesperidin reference substance, and serial numbers 2-4 are bergamot formula granules (the batch numbers of bergamot slices are 20191012, 190102A50, and 190903A50, respectively). Figure 8 Serial number 1 is the 5,7-dimethoxycoumarin reference substance, and serial numbers 2-4 are bergamot formula granules (the batch numbers of bergamot slices are 20191012, 190102A50, and 190903A50, respectively).

[0102] The thin layer identification of the Chinese Pharmacopoeia (2020 Edition) and the National Drug Standards for Chinese Herbal Formula Granules (First Batch) uses bergamot as a reference material for identification, but lacks comparison with reference materials. Compared with the method of using bergamot as a reference material for comparison, this method has strong specificity and good repeatability.

[0103] Example 4

[0104] Experimental study on the content determination of bergamot formula granules by HPLC

[0105] 1. Instruments and test drugs

[0106] 1.1 Instrument

[0107] Table 5 Experimental instruments and equipment

[0108]

[0109] 1.2 Reagents

[0110] Table 6 Reagents and materials

[0111]

[0112] 1.3 Buddha's Hand Pieces

[0113] Table 7 Sample information

[0114]

[0115] 2. Methods and Results

[0116] 2.1 Preparation of reference solution:

[0117] Accurately weigh appropriate amounts of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin reference substances, dissolve them in methanol, make up to volume, and shake well to obtain reference substance stock solutions with concentrations of 857.5, 655.2, 698.7, 816.6, and 502.0 μg / mL, respectively.

[0118] 2.2 Preparation of test solution:

[0119] Accurately weigh 0.2 g of the bergamot formula granules (Example 2), place in a stoppered conical flask, add 20 ml of methanol, weigh, treat with ultrasound (frequency 40 KHz) for 30 minutes, cool, make up the weight loss with methanol, filter, and take the filtrate to obtain the bergamot formula granules test solution;

[0120] Accurately weigh 5 g of bergamot slices, place in a round-bottom flask, add 50 ml of 70% ethanol solution, reflux and extract twice, each time for 1.5 h, filter, and take the filtrate to obtain the 70% ethanol extract of bergamot for testing solution.

[0121] 2.3 Chromatographic conditions

[0122] 2.3.1 Detector Optimization

[0123] Existing literature reports the use of ELSD to determine triterpenoid components, but the ELSD detector has large baseline noise, low sensitivity, and narrow linear range. CAD is a new type of universal detector developed in recent years. It has been used in the analysis of terpenoid components. Studies have shown that when CAD and ELSD detectors are used to detect the content of terpenoid lactones, the CAD detector has higher sensitivity. The three components of diosmin, hesperidin and 5,7-dimethoxycoumarin have strong absorption under the DAD detector, and the chromatogram baseline is stable at a wavelength of 254nm. Compared with the CAD detector, the response is better and the sensitivity is higher; limonin and nomilin have weak absorbance at a wavelength of 254nm and strong absorbance at a wavelength of 210nm, but the chromatogram baseline drift is large, while the signal is stronger under the CAD detector, and the sensitivity is much greater than that of the DAD detector. Therefore, this experiment uses the DAD detector to detect diosmin, hesperidin and 5,7-dimethoxycoumarin at a wavelength of 254nm, and uses the CAD detector to detect limonin and nomilin, so that no interference is achieved, the baseline is relatively stable, and the effect of separation and analysis can be quickly achieved.

[0124] 2.3.2 Optimization of elution conditions

[0125] According to Table 8, the bergamot formula granule test solution was gradient eluted (using DAD detector, 254nm), and the results are shown in Fig. 9 The results showed that the three components of diosmin, hesperidin and 5,7-dimethoxycoumarin had strong absorption under the DAD detector, and the chromatogram baseline was stable at a wavelength of 254nm, with good response and high sensitivity; limonin and nomilin had weak absorbance at a wavelength of 254nm and no obvious peaks.

[0126] Table 8 Gradient elution conditions

[0127]

[0128]

[0129] According to the results of gradient elution in Table 8, the gradient of the Buddha's Hand Formula Granules was optimized. Since the phosphoric acid in the 0.05% phosphoric acid aqueous solution is non-volatile, it will remain in the CAD detector, resulting in contamination of the CAD detector and affecting the normal use and detection results of the CAD detector. Therefore, 0.1% formic acid water was used as the mobile phase. The results showed that the chromatogram obtained under the gradient elution conditions of Table 9 had the best separation effect and significantly shortened the elution time. The chromatogram is shown in FIG. Fig.10 .

[0130] Table 9 Gradient elution conditions

[0131]

[0132] 2.3.3 Screening of mobile phase

[0133] The inventors compared the separation effects of four mobile phase systems, methanol-pure water, acetonitrile-pure water, methanol-0.1% formic acid aqueous solution, and acetonitrile-0.1% formic acid aqueous solution, on the five index components in bergamot according to the gradient elution conditions in Table 9. The results are shown in Table 9. Fig.11 The results showed that when methanol-pure water was used as the mobile phase system, no chromatographic peaks were observed; when methanol-0.1% formic acid water was used as the mobile phase system, the elution ability was weak, and the five index components could not be effectively separated under the gradient conditions; when acetonitrile-pure water was used as the mobile phase system, there were problems such as delayed peak time and wide chromatographic peaks; when acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase system, the chromatogram baseline was stable, the chromatographic peaks were sharp, and the five index components could be effectively separated, so acetonitrile-0.1% formic acid aqueous solution was selected as the mobile phase system.

[0134] 2.3.4 Final chromatographic conditions

[0135] Chromatographic column: Thermo Hypersil GOLD C 18 (250mm×4.6mm,5μm); mobile phase: 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution (0-8min, 20%B; 8-10min, 20%-30%B; 10-15min, 30%B; 15-17min, 30%-53%B, 17-25min, 53%B); flow rate: 1ml / min; column temperature: 30℃; injection volume: 10μL; detectors include DAD and CAD, among which the detection wavelength of DAD detector is 254nm; CAD detector atomization temperature: 35℃, acquisition frequency: 10Hz, filtration constant: 5.0s.

[0136] 2.4 Methodological Review

[0137] 2.4.1 Investigation of linear relationship

[0138] 1 ml of each reference substance stock solution was taken and placed in a 10 ml volumetric flask, and methanol was used to make up to volume to obtain a mixed standard solution. The mixed standard solution was diluted 2, 4, 8, and 10 times in sequence to prepare reference substance solutions of different concentrations. The standard curve was drawn according to the above chromatographic conditions with the injection volume as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 10. The r values ​​were all greater than 0.999, indicating a good linear relationship.

[0139] Table 10 Linear relationships of 5 reference substances

[0140]

[0141] 2.4.2 Precision investigation

[0142] The same mixed reference solution was taken and measured 6 times continuously under the above chromatographic conditions, and the peak areas of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin were recorded and the RSD values ​​were calculated. The results are shown in Table 11. The RSD values ​​were 0.66%, 0.27%, 1.18%, 0.86%, and 0.65%, respectively, indicating that the instrument had good precision.

[0143] Table 11 Precision investigation

[0144]

[0145]

[0146] 2.4.3 Repeatability study

[0147] Six portions of the same batch of bergamot formula granules were accurately weighed, and the test solution was prepared according to the above method. The sample was injected and analyzed, and the content (mg / g) and RSD values ​​of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin were calculated. The results are shown in Table 12. The RSD values ​​were 1.96%, 1.86%, 1.24%, 0.82%, and 0.95%, respectively, indicating that the method had good repeatability.

[0148] Table 12 Repeatability study

[0149]

[0150] 2.4.4 Stability study

[0151] 0.2 g of bergamot formula granules were accurately weighed, and the test solution was prepared according to the above method. The samples were injected and analyzed at 0, 2, 4, 8, 12, and 24 hours, and the contents (mg / g) and RSD values ​​of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin were calculated. The results are shown in Table 13. The RSD values ​​were 0.49%, 0.77%, 1.27%, 1.65%, and 1.37%, respectively, indicating that the test solution had good stability within 24 hours.

[0152] Table 13 Stability Study

[0153]

[0154] 2.4.5 Investigation of sample recovery rate

[0155] Weigh 0.1 g of bergamot formula granules accurately, and add 5 component reference substances in 6 parallel portions. Prepare the test solution according to the above method, inject and analyze, and the experimental results are shown in Table 14. The average recoveries of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin were 99.47%, 96.47%, 94.68%, 95.77%, and 102.17%, respectively, and the RSD values ​​were 1.01%, 1.00%, 1.50%, 1.85%, and 1.43%, respectively, indicating that the method has high accuracy.

[0156] Table 14 Sample recovery results

[0157]

[0158] 2.5 Transfer Rate

[0159] According to the content of each component in the bergamot slices in Table 15 and the content of each component in the bergamot formula granules in Table 17, the transfer rate of each component was calculated. The results are shown in Table 16. The calculation formula is as follows:

[0160]

[0161] Wherein c0 and c1 are the contents of individual components in 70% ethanol extract of bergamot and bergamot formula granules, respectively, m0 is the mass of bergamot slices when preparing bergamot formula granules, and m1 is the mass of the prepared bergamot formula granules.

[0162] Table 15 Determination results of different components in different batches of 70% ethanol extracts of bergamot (mg / g)

[0163]

[0164] Table 16 Transfer rate of different batches of bergamot particles (%)

[0165]

[0166] 2.6 Sample determination

[0167] Three batches of bergamot slices were taken respectively, and the formula granules and test solutions were prepared according to the above method. The contents (mg / g) of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin and nomilin were calculated. The results are shown in Table 17.

[0168] Table 17 Determination results of the contents of five components in different batches of bergamot granules (mg / g)

[0169]

[0170] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing bergamot formula granules, characterized in that: The steps include: (1) extracting the bergamot slices twice with alcohol, filtering, combining the filtrates, concentrating under reduced pressure until there is no alcohol taste, and adding water to obtain a concentrated solution; (2) passing the concentrated solution through a macroporous adsorption resin column for adsorption, eluting with alcohol, and collecting the eluate; (3) The eluate is concentrated under reduced pressure to an extract, auxiliary materials are added and mixed evenly, and the extract is granulated, dried, and sized to obtain the bergamot formula granules.

2. The method for preparing the bergamot formula granules according to claim 1, characterized in that: In step (1), the alcohol extraction conditions are: reflux extraction twice with 70% ethanol in a volume 8-12 times of the volume of the bergamot slices, each time for 1-2 hours; and adding water to a crude drug concentration of 0.4-0.6 g / ml.

3. The method for preparing the bergamot formula granules according to claim 1, characterized in that: In step (2), the model of the macroporous adsorption resin column is AB-8, the mass ratio of the bergamot slices to the resin is 1:1.8-4, the column is wet packed with water, the diameter-to-height ratio is 1:3-5, and the adsorption time is 15-30 minutes; The alcohol elution condition is: washing with 7-10 column volumes of water until the effluent has no Molish reaction, and then eluting with 5-6 column volumes of 70% ethanol.

4. The method for preparing the bergamot formula granules according to claim 1, characterized in that: In step (3), 0.2-0.3 times the mass of dextrin of the bergamot slices is added and mixed evenly, 0.1-0.2 times the mass of 70% ethanol of the bergamot slices is used as a wetting agent for wet granulation, granulation is carried out through a No. 1 sieve, and drying is carried out at 50-60° C. for 60-90 minutes. Granules that can pass through the No. 1 sieve and cannot pass through the No. 5 sieve are defined as qualified granules, thereby obtaining bergamot formula granules.

5. A bergamot formula granule, characterized in that: The method is prepared by the method according to any one of claims 1 to 4.

6. A quality control method for the bergamot formula granules according to claim 5, characterized in that: include: A step of performing thin layer chromatography to identify hesperidin and 5,7-dimethoxycoumarin in the bergamot formula granules; as well as, The invention relates to a step of determining the contents of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin and nomilin in the bergamot formula granules by high performance liquid chromatography.

7. The quality control method of the bergamot formula granules according to claim 6, characterized in that: The step of thin layer chromatography identification comprises: (1) Preparation of test solution: 0.2 g of the bergamot formula granules were added with 20 ml of methanol, ultrasonicated, cooled, filtered, the filtrate was evaporated to dryness, and 1 ml of methanol was added to the residue as the test solution; (2) Preparation of reference solution: Take hesperidin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the hesperidin reference substance solution; take 5,7-dimethoxycoumarin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the 5,7-dimethoxycoumarin reference substance solution; (3) Determination: 2-10 μl of the test solution and hesperidin reference solution were taken and spotted on the same silica gel plate, respectively, and the lower layer solution of chloroform-ethyl acetate-methanol-water in a volume ratio of 3:8:4:2 was used as the developing solvent. The plate was taken out, dried, sprayed with 1% aluminum chloride ethanol solution, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference substance. Pipette 2-10 μl of the test solution and 5,7-dimethoxycoumarin reference solution, respectively, and spot them on the same silica gel plate. Use n-hexane-acetone-chloroform in a volume ratio of 10:6:1 as the developing solvent. Develop the plate, take out, dry it, and examine it under a 365nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference substance.

8. The quality control method of the bergamot formula granules according to claim 7, characterized in that: In step (3), the spotting volume of the test solution and the hesperidin reference solution is 2 μl; The silica gel plate is a hand-laid plate or a silica gel G plate, and the unfolding environment is: temperature 10-26° C., relative humidity 32.0%-72.0%.

9. The quality control method of the bergamot formula granules according to claim 6, characterized in that: The step of high performance liquid chromatography assay comprises: (1) Preparation of mixed reference substance solution: accurately weigh appropriate amounts of diosmin, hesperidin, 5,7-dimethoxycoumarin, limonin, and nomilin reference substances, dissolve them in methanol, make up to volume, and shake well to obtain reference substance stock solutions with concentrations of 857.5, 655.2, 698.7, 816.6, and 502.0 μg / ml, respectively; Take 1 ml of each reference substance stock solution and place it in a 10 ml volumetric flask, add methanol to make up to volume to obtain a mixed standard solution; The mixed standard solution was diluted 2, 4, 8, and 10 times by the multiple dilution method to prepare mixed reference solutions of different concentrations; (2) Preparation of the test solution: accurately weigh 0.2 g of the bergamot formula granules, place in a stoppered conical flask, add 20 ml of methanol, weigh, ultrasonicate, cool, add methanol to make up the weight loss, filter, and take the filtrate to obtain the test solution; (3) Determination: 2-10 μl of the test solution and the mixed reference solution were respectively taken and injected into a liquid chromatograph for determination. The chromatographic conditions were: the chromatographic column was C 18 The filler was octadecylsilane bonded silica gel, with 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution: 0-8min, 20% B; 8-10min, 20%-30% B; 10-15min, 30% B; 15-17min, 30%-53% B, 17-25min, 53% B, the flow rate was 1ml / min, the column temperature was 30℃, and the liquid chromatograph detectors were DAD and CAD.

10. The quality control method of the bergamot formula granules according to claim 9, characterized in that: In step (3), the detection wavelength of the DAD detector is 254 nm; the atomization temperature of the CAD detector is 35° C., the acquisition frequency is 10 Hz, and the filtration constant is 5.0 s.

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