A quality detection method for salt orange seed formula granules

Abstract

The present invention provides a salt orange core formula granule quality detection method, including through the properties of the salt orange core formula granule, dry extract paste rate, thin layer identification, extract, characteristic spectrum and limonin, nomilin, and total content determination of flavonoids, the salt orange core formula granule content standard is limited to 8.7-18.8mg per 1g of limonin, nomilin, and total content of flavonoids, wherein the thin layer identification is identified by thin layer chromatography; the extract is determined by hot dipping; the characteristic spectrum and limonin, nomilin, and total content determination of flavonoids are all determined by liquid chromatography. The salt orange core formula granule quality detection method of the present invention, by systematic detection to evaluate the quality of the salt orange core formula granule, can comprehensively reflect the intrinsic quality of the formula granule and better control the quality of the salt orange core formula granule.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a quality detection method for salt orange seed formula granules. Background Art

[0002] Modern drugs need to have three characteristics: stability, uniformity, safety and effectiveness. For Chinese patent medicines, multiple means are needed to ensure the reliability and stability of the test results. Chinese medicine formula granules are single-flavor Chinese medicine products made by extracting, concentrating, drying and other processes of Chinese medicine slices using modern scientific and technological methods, imitating the traditional Chinese medicine decoction decoction method. The product maintains the nature, taste and efficacy of Chinese medicine slices, has stable quality, and is used in the preparation of Chinese medicine clinical prescriptions, adapting to the needs of syndrome differentiation and treatment, and prescription changes. It has the advantages of no need for decoction, easy to take, fast absorption, accurate dosage, safe and clean, and easy to carry. The single-flavor medicinal material is obtained by water extraction, concentration, drying, granulation and other processes to obtain formula granules. Compared with traditional medicinal materials, the morphological characteristics of the formula granules have changed significantly, and the authenticity and quality of the product cannot be observed with the naked eye. Therefore, it is necessary to establish a quality evaluation system for Chinese medicine formula granules to comprehensively reflect the intrinsic quality of the formula granules. Salt orange core formula granules are formula granules made from dried mature seeds of the Rutaceae plant Citrus reticulata Blanco and its cultivated varieties, which are processed and processed according to the main quality standards of standard decoctions. They are bitter, flat, non-toxic, enter the liver and kidney meridians, regulate qi, relieve pain, and are mainly used to treat hernia, testicular swelling and pain, mastitis, low back pain, and bladder gas pain. At present, there is no systematic quality detection method for the formula granules of salt orange core. It is not comprehensive enough to use only existing detection methods to detect the salt orange core formula granules, which cannot reflect the overall intrinsic quality of the salt orange core formula granules and cannot meet the quality control requirements of traditional Chinese medicine formula granules. Therefore, it is very necessary to establish a quality detection method for salt orange core formula granules for medicinal material quality control. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies of the prior art and to provide a method for detecting the quality of salt orange seed formula particles, which can comprehensively reflect the intrinsic quality of the formula particles and better control the quality of the formula particles.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] A method for detecting the quality of salt orange seed formula particles, comprising the following detection method:

[0006] Through the properties of salt orange seed formula granules, dry extract yield, thin layer identification, characteristic spectrum, extract and total content of limonin, nomilin and flavone, the content standard of the formula granules is limited to 8.7-18.8 mg of limonin, nomilin and flavone per 1g. Among them, thin layer identification is carried out by thin layer chromatography; characteristic spectrum and total content of limonin, nomilin and flavone are determined by liquid chromatography; extract is determined by hot immersion method;

[0007] The characteristic spectrum is determined by liquid chromatography, including: performing liquid chromatography analysis, using a solution made from a tangerine seed control medicinal material as a control medicinal material reference solution b, using a solution made from a limonin and hesperidin reference substance as a reference substance reference solution b, using a solution made from a salt tangerine seed formula granule sample as a test solution b, accurately aspirating the control medicinal material reference solution b, the reference substance reference solution b and the test solution b, respectively, injecting them into a liquid chromatograph, and determining, to obtain; wherein the chromatographic conditions used are: a chromatographic column: using octadecylsilane bonded silica gel as a filler, a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; a mobile phase: using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the provisions of Table a;

[0008] Table a Gradient elution program

[0009] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～3 20 80 3～18 20→23 80→77 18～19 23→23.5 77→76.5 19～25 23.5→24 76.5→76 25～28 24 76 28～34 24→36 76→64 34～49 36→41 64→59 49～55 41→43 59→57 55～65 43→47 57→53 65～87 47→100 53→0 87～97 100 0 97～99 100→20 0→80

[0010] Flow rate: 1.2 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 220 nm.

[0011] In one embodiment, the preparation method of the salt orange seed formula granules is as follows: take 4500g of salt orange seed slices, add water to boil, filter, concentrate the filtrate into a clear paste, add granule excipients, dry or dry and crush, then add granule excipients, mix well, granulate, and make 1000g.

[0012] In one embodiment, the thin layer chromatography method comprises the following steps: (1) preparing a test solution a: taking 1.0 g of a salt orange seed formula granule sample, adding 20 mL of petroleum ether, ultrasonically treating for 30 min, filtering, discarding the filtrate, adding 20 mL of dichloromethane to the residue, ultrasonically treating for 20 min, filtering, evaporating the filtrate, and adding 1 mL of methanol to the residue to dissolve it, thereby preparing a test solution a; (2) preparing a control medicinal material solution a: taking 1.0 g of an orange seed control medicinal material, adding 20 mL of petroleum ether, ultrasonically treating for 30 min, filtering, discarding the filtrate, adding dichloromethane to the residue, and dissolving it. 20ml, ultrasonic treatment for 20min, filtration, evaporation of the filtrate, and dissolution of the residue in 1ml of methanol to prepare a control medicinal solution a; (3) thin layer chromatography analysis: thin layer chromatography conditions are as follows: thin layer plate: silica gel G thin layer plate; sample volume: 4ul of test solution a and 8ul of control medicinal solution a; developing agent: dichloromethane-ethyl acetate solution with a volume ratio of 4:1 as the developing agent; color developer: dimethylbenzylidene rhodanine-sulfuric acid-ethanol solution with a mass percentage of 1:2:97, heated at 105°C, and inspected under ultraviolet light at 365nm.

[0013] In one embodiment, the petroleum ether is 60-90°C petroleum ether.

[0014] In one embodiment, the hot dipping method uses ethanol as solvent, and the hot dipping method under the alcohol-soluble extract determination method is used to determine the extract range.

[0015] In one embodiment, the characteristic spectrum is determined by liquid chromatography and further comprises the following steps: (1) preparing a reference medicinal material reference solution b: taking 0.5 g of orange seed reference medicinal material, adding 15 mL of methanol, reflux for 50 min, cooling, filtering, and taking the filtrate as the reference medicinal material reference solution b; (2) preparing a reference substance reference solution b: taking an appropriate amount of limonin and hesperidin reference substances, accurately weighing, adding methanol to prepare a mixed solution containing 500 μg of limonin and 10 μg of hesperidin per 1 ml, as the reference substance reference solution b; (3) preparing a test solution b: taking an appropriate amount of salt orange seed formula granules, grinding, taking about 0.4 g, placing in a stoppered conical flask, adding 15 mL of methanol, heating and reflux for 30 min, cooling, shaking, filtering, and taking the filtrate as the test solution b.

[0016] In one embodiment, the total content of limonin, nomilin and phedrine is determined by liquid chromatography, including: performing liquid chromatography analysis, using a solution made from limonin, nomilin and phedrine reference substances as the reference solution c, and using a solution made from a salt orange seed formula granule sample as the test solution c, accurately aspirating the reference solution c and the test solution c respectively, injecting them into the liquid chromatograph respectively, and determining to obtain the result; wherein, the chromatographic conditions used are: chromatographic column: octadecylsilane bonded silica gel as the filler, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; mobile phase: acetonitrile-water with a volume ratio of 35:65 as the mobile phase; flow rate: 1.0 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 220 nm.

[0017] In one embodiment, the total content of limonin, nomilin and phedrine is determined by liquid chromatography, which further comprises the following steps: (1) preparing a reference solution: taking appropriate amounts of limonin, nomilin and phedrine reference substances, accurately weighing them, and adding methanol to prepare a mixed solution containing 300 μg of limonin, 200 μg of nomilin and 50 μg of phedrine per 1 ml, as the reference solution c; (2) preparing a test solution: taking appropriate amounts of salt orange seed formula granules, grinding them into powder, taking about 0.2 g, accurately weighing it, placing it in a stoppered conical flask, accurately adding 15 ml of methanol, sealing it, weighing it, heating it under reflux for 20 min, cooling it, weighing it again, making up the lost weight with methanol, shaking it well, filtering it, and taking the filtrate as the test solution c.

[0018] Compared with the prior art, the quality detection method of the salt orange core formula granules provided by the present invention has the following beneficial effects: by studying the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and total content determination of limonin, nomilin and flavonoids of the salt orange core formula granules, the quality of the salt orange core formula granules is evaluated through multi-faceted measurements, laying a solid foundation for the stable quality of the product, and being able to establish a feasible quality standard for the salt orange core formula granules, and realizing effective control of the quality of the salt orange core formula granules. Moreover, by using the chromatographic conditions of the present application for liquid phase analysis, a characteristic spectrum with better separation and clearer can be obtained, and the method has good stability, high precision and good reproducibility. The salt orange core decoction pieces are decocted to obtain the salt orange core decoction pieces formula granules, and the total content of limonin, nomilin and flavonoids is 12.49 mg / g on average. According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", the total content of limonin, nomilin and flavonoids is set to be 8.7-18.8 mg / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The TLC spectra of three batches of production samples of the salt orange core decoction granules according to thin layer chromatography in one embodiment of the present invention; among them, Group 1 is a negative control 4μl, Group 2 is a control medicinal material solution 8μl, Group 3 is a test solution 4μl, Group 4 is a test solution 4μl, and Group 5 is a test solution 4μl.

[0020] Figure 2 The HPLC characteristic spectra of different extraction methods in the investigation of the characteristic spectrum determination extraction method of one embodiment of the present invention are shown; wherein S1 is the characteristic spectrum of the test solution extracted by ultrasonic extraction; and S2 is the characteristic spectrum of the test solution extracted by reflux extraction.

[0021] Figure 3 This is the HPLC characteristic spectrum of different extraction solvents in the characteristic spectrum determination extraction solvent investigation of one embodiment of the present invention.

[0022] Figure 4 This is a HPLC characteristic spectrum of different extraction times in the characteristic spectrum determination extraction time investigation of one embodiment of the present invention.

[0023] Figure 5 The HPLC characteristic spectra of different sample amounts in the characteristic spectrum determination sample amount investigation of one embodiment of the present invention; wherein S1 is the characteristic spectrum of the test solution with a sample amount of 1.0 g; S2 is the characteristic spectrum of the test solution with a sample amount of 0.4 g; and S3 is the characteristic spectrum of the test solution with a sample amount of 0.6 g.

[0024] Figure 6 The HPLC characteristic spectra of different solutions in the investigation of the specificity of the characteristic spectrum determination of one embodiment of the present invention are shown; wherein S1 is the characteristic spectrum of the maltodextrin blank control reference, S2 is the characteristic spectrum of the mixed reference reference, S3 is the characteristic spectrum of the tangerine seed control medicinal material reference, and S4 is the characteristic spectrum of the salt tangerine seed test solution.

[0025] Figure 7 This is a common peak overlay spectrum of a repeatability test in a characteristic spectrum determination according to an embodiment of the present invention.

[0026] Figure 8 This is a common peak overlay spectrum of the precision test in the characteristic spectrum determination of one embodiment of the present invention.

[0027] Fig. 9 This is an overlay spectrum of common peaks in a stability test in the characteristic spectrum determination according to one embodiment of the present invention.

[0028] Fig.10 This is a characteristic spectrum of an embodiment of the present invention, in which different column temperatures (±5°C) are examined and superimposed on each other; wherein S1 is a chromatographic column with a column temperature of 25°C, S2 is a chromatographic column with a column temperature of 30°C, and S3 is a chromatographic column with a column temperature of 35°C.

[0029] Fig.11 This is a characteristic spectrum of a common peak superposition characteristic spectrum of a characteristic spectrum determination flow rate test (±0.2ml / min) of an embodiment of the present invention; wherein S1 is a chromatographic column with a flow rate of 1.0ml / min, S2 is a chromatographic column with a flow rate of 1.2ml / min, and S3 is a chromatographic column with a flow rate of 1.4ml / min.

[0030] Fig.12 This is an overlay of characteristic spectra of three batches of finished products in one embodiment of the present invention; among them, the finished product number of S1 is 221201, the finished product number of S2 is 221202, and the finished product number of S3 is 221203.

[0031] Fig.13 This is an overlay diagram of the finished product of salt orange seeds, the reference substance, and the reference medicinal material in one embodiment of the present invention.

[0032] Fig.14 This is a characteristic spectrum fitting diagram of the salt orange seed finished product solution and the orange seed control medicinal material solution in one embodiment of the present invention; wherein, peak 1 (S1) is hesperidin; peak 7 (S2) is limonin; peak 9 is nomiline; peak 10 is phellodendron; the chromatographic column used is SPOLAR C18-AQ, 4.6mm×250mm, 5μm.

[0033] Fig.15 This is a comparison chart of maltodextrin blanks in the investigation of content determination specificity in one embodiment of the present invention; wherein, S1 is a maltodextrin blank, S2 is a mixed reference substance of limonin, nomilin, and chloroquine, and S3 is a salt orange core test substance. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0035] The invention provides a quality detection method for salt orange seed formula granules, comprising the following detection method: by determining the properties, dry extract yield, thin layer identification, characteristic spectrum, extract and total content of limonin, nomilin and flavone of the salt orange seed formula granules, the content standard of the formula granules is limited to 8.7-18.8 mg of total content of limonin, nomilin and flavone per 1g, wherein the thin layer identification is performed by thin layer chromatography; the characteristic spectrum and the total content of limonin, nomilin and flavone are both determined by liquid chromatography; the extract is determined by hot immersion method.

[0036] In this embodiment:

[0037] Preparation of salt orange core formula granules: Formula granules made according to the "technical requirements" of the source and medicinal parts of the Chinese Pharmacopoeia 2020, including the family name, Chinese name, Latin and medicinal parts of the medicinal plant, that is, this product is a formula granule made from the dried mature seeds of the Rutaceae plant Citrus reticulata Blanco and its cultivated varieties, which are processed and processed according to the main quality standards of the standard decoction. The specific preparation method is: take 4500g of salt orange core slices, add water to decoct, filter, concentrate the filtrate into a clear paste (the dry extract yield is 11.3% to 20.5%), add an appropriate amount of auxiliary materials, dry (or dry, crush), add an appropriate amount of auxiliary materials, mix, granulate, and make 1000g. According to the yield of 21 batches of standard decoctions, the dry extract yield is determined as the dry extract yield in the range of 70% to 130% of its average, and the dry extract yield is 11.3% to 20.5%.

[0038] 1. Characteristic investigation

[0039] Properties: Take one sample each from three batches of large-scale production of salt orange seed granules, observe and record the morphology, color and smell of the samples, and combine the color and smell of the standard soup samples. This product is a granule, a light yellow to brown powder, with a slight odor and bitter taste.

[0040] 2. Thin layer identification

[0041] With reference to the method of the published document "Wang Xiaoqing. Research on the processing technology and quality standard of salt orange kernel and vinegar myrrh [D] Guangdong Pharmaceutical College. 2015", the thin layer identification method of this product was established with orange kernel as the control medicinal material. After methodological investigation and 26 batches of sample tests, the test sample spots were clear and the negative control sample had no interference. Therefore, this method was included in the [Identification] item of the quality standard of salt orange kernel formula granules. Specifically, the thin layer identification method of salt orange kernel formula granules is:

[0042] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0 g, add 20 ml of petroleum ether (60-90°C), ultrasonically treat it for 30 minutes, filter, discard the filtrate, add 20 ml of dichloromethane to the residue, ultrasonically treat it for 20 minutes, filter, evaporate the filtrate, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0043] Control medicinal material solution: Take another 1.0 g of orange seed control medicinal material and prepare the control medicinal material solution in the same way.

[0044] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 4 μl of the test solution and 8 μl of the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively, with dichloromethane-ethyl acetate (4:1) solution as the developing solvent, developed, taken out, dried, sprayed with dimethylbenzylidene rhodanine: sulfuric acid: ethanol = 1:2:97 (W / W) solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material.

[0045] According to the above detection method, take three batches of production sample test solution, negative sample (maltodextrin) solution and control medicinal material solution, spot and perform thin layer identification. Figure 1 As shown, the results show that in the chromatograms of the three batches of large-scale production samples, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal materials.

[0046] 3. Feature Spectrum

[0047] 3.1 Characteristic spectrum determination method

[0048] 3.1.1 Chromatographic conditions and system suitability experiments Octadecylsilane bonded silica gel was used as filler (SPOLARC18 S5 (4.6 mm × 250 mm, 5 μm)); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 1.2 ml per minute; the column temperature was 30°C; the detection wavelength was 220 nm. The injection volume was 10 μl. The number of theoretical plates calculated based on the limonin peak should not be less than 5000.

[0049] Table 1 - Gradient elution program

[0050] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～3 20 80 3～18 20→23 80→77 18～19 23→23.5 77→76.5 19～25 23.5→24 76.5→76 25～28 24 76 28～34 24→36 76→64 34～49 36→41 64→59 49～55 41→43 59→57 55～65 43→47 57→53 65～87 47→100 53→0 87～97 100 0 97～99 100→20 0→80

[0051] 3.1.2 Preparation of reference solution

[0052] Take 0.5 g of orange seed control medicinal material, add 15 ml of methanol, reflux for 50 minutes, cool, filter, and take the filtrate as the control medicinal material reference solution.

[0053] Take appropriate amount of limonin and hesperidin reference substances, weigh accurately, add methanol to prepare mixed solutions containing 500 μg of limonin and 10 μg of hesperidin per 1 ml, as reference substance solutions.

[0054] 3.1.3 Preparation of test solution

[0055] Take an appropriate amount of this product, grind it into powder, take about 0.6g, put it in a stoppered conical flask, add 15ml of methanol, weigh the weight, heat and reflux for 40 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0056] 3.1.4 Determination method

[0057] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure to obtain the result.

[0058] 3.2 Methodological investigation of characteristic spectrum determination

[0059] The sample pretreatment methods of the characteristic spectra of salt orange seed formula granules were investigated, mainly focusing on the effects of extraction method, extraction solvent, extraction time and sampling amount on the characteristic spectra of salt orange seed formula granules.

[0060] 3.2.1 Investigation of extraction methods

[0061] Prepare the test sample solution using different extraction methods and perform the test according to the method in “3.1” above. Figure 2 As shown in Table 2, the results show that the number of main peaks of the chromatographic solution of the test sample with different extraction methods is consistent, and the peak shape difference is not obvious, but the reflux extraction effect is better, so reflux is selected as the sample extraction method.

[0062] Table 2 - Comparison of extraction methods

[0063]

[0064] 3.2.2 Investigation of extraction solvent

[0065] Prepare the test sample solution with different extraction solvents and perform the determination according to the method under "3.1" above. Figure 3 As shown in Table 3, the results show that the number of main peaks in the chromatograms of the test sample solutions with different extraction solvents is consistent, and the peak shapes are not significantly different, but methanol extraction has a better effect, so methanol is selected as the test sample extraction solvent.

[0066] Table 3 - Comparison of extraction solvents

[0067]

[0068] 3.2.3 Extraction time investigation

[0069] Prepare the test solution at different times and perform the test according to the method under "3.1" above. Figure 4 As shown in Table 4, the results show that the number of main peaks in the chromatogram of the test solution at different extraction times is consistent, the peak shape is not significantly different, and the extraction effect is not much different. In order to save time, the extraction time of the sample is determined to be 30 minutes.

[0070] Table 4 - Comparison of extraction times

[0071]

[0072] 3.2.4 Investigation of sampling volume

[0073] Prepare the test sample solution with different sampling volumes and perform the determination according to the method under "3.1" above. Figure 5 As shown in Table 5, the results show that the number of main peaks of the chromatograms of the test solution extracted with different sampling amounts is consistent, and the peak shapes are slightly different, but the sample solution with a sampling amount of 0.4 g has a better extraction effect, so 0.4 g is selected as the sampling amount for the preparation of the test solution.

[0074] Table 5 - Comparison of sampling volumes

[0075]

[0076] In summary, the method for preparing the test solution is as follows: take an appropriate amount of the product, grind it into powder, take about 0.4 g, put it in a stoppered conical flask, add 15 ml of methanol, reflux for 30 minutes, cool and shake well, filter, and take the filtrate to obtain the solution.

[0077] 3.3 Verification of characteristic spectrum analysis method

[0078] 3.3.1 Specificity inspection

[0079] Take an appropriate amount of maltodextrin and prepare a maltodextrin blank control solution according to the method under "3.1" above. Prepare a salt orange seed control medicinal material solution and a mixed solution of limonin and hesperidin reference substances according to the method under "3.1" above. Determine according to the chromatographic conditions under "3.1" above.

[0080] The results showed that the main characteristic peaks in the chromatogram of the test sample were well separated, and the maltodextrin blank had no interference. The method had good specificity. Figure 6 .

[0081] 3.3.2 Repeatability test

[0082] Take about 0.4g of salt orange seed formula granules (batch number: 221103), a total of 6 portions, and perform the test according to the method under "3.1". Figure 7 As shown, the peak shape and number of peaks in the characteristic spectrum of the test sample are basically consistent. There are 10 common peaks in the characteristic spectrum. The peak corresponding to the hesperidin reference peak is S1. The relative retention time and relative peak area of ​​peak 2, peak 3, peak 4, peak 5, peak 6 and S1 are calculated. The peak corresponding to the limonin reference peak is S2. The relative retention time and relative peak area of ​​peak 8, peak 9, peak 10 and S2 are calculated, and the RSD% value is calculated. The calculation results show that the relative peak area RSD% value and the relative retention time RSD% value are both within the qualified range, as shown in Table 6-7. The experiment shows that the method has good repeatability.

[0083] Table 6 - Relative retention time of HPLC characteristic spectrum in repeatability test

[0084] Peak 1 2 3 4 5 6 RSD(%) 1(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 2 1.153 1.152 1.153 1.155 1.156 1.155 0.12 3 1.520 1.518 1.521 1.519 1.519 1.519 0.06 4 1.621 1.619 1.624 1.626 1.626 1.625 0.18 5 1.712 1.709 1.713 1.709 1.709 1.711 0.10 6 1.962 1.958 1.964 1.960 1.959 1.959 0.11 7(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 8 1.036 1.036 1.036 1.035 1.035 1.036 0.03 9 1.126 1.126 1.126 1.126 1.126 1.126 0.02 10 1.277 1.277 1.277 1.276 1.277 1.277 0.03

[0085] Table 7 - Relative peak area of ​​HPLC characteristic spectrum of repeatability test

[0086]

[0087]

[0088] 3.3.3 Precision test

[0089] Take about 0.4g of the salt orange core formula granules (batch number: 221103), a total of 6 samples, and perform 6 consecutive injections for determination according to the test method under "3.1". The measurement results show that the peak shape and number of peaks in the characteristic spectrum of the test sample are basically consistent. There are 10 common peaks in the characteristic spectrum. The peak corresponding to the hesperidin reference peak is S1. The relative retention time and relative peak area of ​​peak 2, peak 3, peak 4, peak 5, peak 6 and peak S1 are calculated. The peak corresponding to the limonin reference peak is S2. The relative retention time and relative peak area of ​​peak 8, peak 9, peak 10 and peak S2 are calculated, and the RSD% value is calculated. The calculation results show that the relative peak area RSD% value and the relative retention time RSD% value are both within the qualified range. For details, see Figure 8 As shown in Table 8-9, the experiment shows that this method has good precision.

[0090] Table 8 - Relative retention time of HPLC characteristic spectrum of precision test

[0091] Peak 1 2 3 4 5 6 RSD(%) 1(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 2 1.153 1.152 1.151 1.150 1.152 1.152 0.08 3 1.515 1.517 1.518 1.517 1.519 1.519 0.10 4 1.620 1.620 1.615 1.613 1.619 1.618 0.18 5 1.702 1.708 1.710 1.708 1.710 1.712 0.19 6 1.954 1.958 1.960 1.956 1.959 1.960 0.12 7(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 8 1.036 1.036 1.037 1.036 1.036 1.036 0.02 9 1.126 1.126 1.126 1.126 1.126 1.126 0.01 10 1.277 1.277 1.277 1.277 1.277 1.277 0.02

[0092] Table 9 - Relative peak area of ​​HPLC characteristic spectrum of precision test

[0093]

[0094]

[0095] 3.3.4 Stability test

[0096] Take about 0.4g of salt orange seed formula granules (batch number: 221103) and test them at 0h, 2h, 4h, 8h, 12h and 24h according to the test method under "3.1". The test results show that the peak shape and peak number of the characteristic spectrum of the test sample are basically consistent (see Fig. 9). There are 10 common peaks in the characteristic spectrum. The peak corresponding to the hesperidin reference peak is S1. The relative retention time and relative peak area of ​​peak 2, peak 3, peak 4, peak 5, peak 6 and S1 are calculated. The peak corresponding to the limonin reference peak is S2. The relative retention time and relative peak area of ​​peak 8, peak 9, peak 10 and S2 are calculated, and the RSD% value is calculated. The calculation results show that the relative peak area RSD% value and the relative retention time RSD% value are both within the qualified range, as shown in Table 10-11. The test shows that the test solution is relatively stable within 24 hours.

[0097] Table 10 - Relative retention time of characteristic spectrum of stability test

[0098] Peak 0h 2h 4h 8h 12h 24h RSD(%) 1(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 2 1.156 1.157 1.156 1.158 1.160 1.161 0.17 3 1.517 1.516 1.516 1.518 1.516 1.513 0.10 4 1.632 1.632 1.632 1.635 1.638 1.638 0.19 5 1.703 1.703 1.704 1.705 1.702 1.697 0.17 6 1.954 1.953 1.954 1.955 1.953 1.948 0.12 7(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 8 1.035 1.035 1.035 1.035 1.035 1.034 0.04 9 1.126 1.126 1.126 1.126 1.126 1.126 0.01 10 1.276 1.277 1.277 1.276 1.276 1.276 0.03

[0099] Table 11 - Relative peak area of ​​characteristic spectrum of stability test

[0100]

[0101]

[0102] 3.4 Durability test

[0103] 3.4.1 Tests at different column temperatures (±5°C)

[0104] Take about 0.4g of the salt orange core formula granules (batch number: 221103) sample and perform the test according to the test method under "3.1" with the column temperatures of 25°C, 30°C and 35°C. There are 10 common peaks in the characteristic spectrum. The peak corresponding to the hesperidin reference peak is S1. The relative retention time and relative peak area of ​​peak 2, peak 3, peak 4, peak 5, peak 6 and peak S1 are calculated. The peak corresponding to the limonin reference peak is S2. The relative retention time and relative peak area of ​​peak 8, peak 9, peak 10 and peak S2 are calculated, and the RSD% value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both out of the qualified range. For details, see Fig.10 Compared with Tables 12-13, the experiment shows that the change of column temperature has a greater impact on the characteristic spectrum determination, that is, the column temperature durability is poor.

[0105] Table 12- Column temperature test HPLC characteristic spectrum relative retention time (±5℃)

[0106] Peak 25℃ 30℃ 35℃ RSD(%) 1(S1) 1.000 1.000 1.000 0.00 2 1.196 1.157 1.105 3.98 3 1.402 1.514 1.364 5.44 4 1.487 1.626 1.537 4.55 5 1.689 1.701 1.617 2.71 6 1.898 1.952 1.969 1.89 7(S2) 1.000 1.000 1.000 0.00 8 1.024 1.035 1.045 1.00 9 1.126 1.126 1.125 0.06 10 1.278 1.276 1.273 0.23

[0107] Table 13- Column temperature test HPLC characteristic spectrum relative peak area (±5℃)

[0108]

[0109]

[0110] 3.4.2 Investigation of different flow rates (±0.2ml / min)

[0111] Take about 0.4g of salt orange core formula granules (batch number: 221103) sample, and measure it according to the test method under "3.1" at flow rates of 1.0ml / min, 1.2ml / min, and 1.4ml / min. There are 10 common peaks in the characteristic spectrum. The peak corresponding to the hesperidin reference peak is S1. The relative retention time and relative peak area of ​​peak 2, peak 3, peak 4, peak 5, peak 6 and peak S1 are calculated. The peak corresponding to the limonin reference peak is S2. The relative retention time and relative peak area of ​​peak 8, peak 9, peak 10 and peak S2 are calculated, and the RSD% value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both out of the qualified range. For details, see Fig.11 The experiment shows that the change of flow rate has a certain influence on the characteristic spectrum determination, that is, the durability of the characteristic spectrum determination is poor under the change of flow rate.

[0112] Table 14-Flow rate test HPLC characteristic spectrum relative retention time (±0.2ml / min)

[0113] Peak 1.0ml / min 1.2ml / min 1.4ml / min RSD(%) 1(S1) 1.000 1.000 1.000 0.00 2 1.149 1.156 1.169 0.88 3 1.491 1.519 1.541 1.64 4 1.605 1.628 1.652 1.43 5 1.659 1.710 1.750 2.67 6 1.896 1.958 2.008 2.87 7(S2) 1.000 1.000 1.000 0.00 8 1.029 1.035 1.040 0.52 9 1.126 1.126 1.124 0.10 10 1.279 1.276 1.272 0.28

[0114] Table 15-Flow rate test HPLC characteristic spectrum relative peak area (±0.2ml / min)

[0115] Peak 1.0ml / min 1.2ml / min 1.4ml / min RSD(%) 1(S1) 1.000 1.000 1.000 0.00 2 0.587 0.573 0.578 1.24 3 0.081 0.069 0.057 17.58 4 0.150 0.154 0.271 35.60 5 0.056 0.068 0.079 17.51 6 0.995 1.001 1.004 0.46 7(S2) 1.000 1.000 1.000 0.00 8 0.054 0.062 0.064 8.52 9 0.083 0.091 0.094 6.56 10 0.340 0.351 0.352 1.87

[0116] 3.3 Summary

[0117] The characteristic spectrum method was tested for specificity, precision, repeatability, stability and durability, and the results met the requirements, indicating that the established method can be well used for the characteristic spectrum determination of salt orange seed formula granules.

[0118] 3.4 Characterization and analysis of characteristic spectra of three batches of finished products

[0119] 3.4.1 Characteristic spectra of three batches of finished products

[0120] According to the characteristic spectrum analysis method proposed above, the characteristic spectrum of three batches of finished products was determined, and the results showed that there were 10 common peaks in the characteristic chromatogram of the formula granules and the Chinese herbal medicine pieces used in their preparation, and the retention times corresponded to the 10 characteristic peaks in the chromatogram of the reference medicinal material reference. Among them, the peak corresponding to the hesperidin reference is peak 1, and the peak corresponding to the limonin reference is peak 7. For details, see Figure 12-14 .

[0121] 3.4.2 Evaluation of characteristic chromatograms of three batches of formulated granules

[0122] According to the characteristic spectrum analysis method proposed above, the characteristic spectrum of three batches of finished products was determined. The results showed that there were 10 common peaks in the characteristic spectrum, and the peak corresponding to the hesperidin reference peak was S1, and the relative retention time of peak 2, peak 3, peak 4, peak 5, peak 6 and S1 was calculated. The peak corresponding to the limonin reference peak was S2, and the relative retention time of peak 8, peak 9, peak 10 and S2 was calculated. The relative retention time and range are shown in Table 16.

[0123] Table 16-3 Relative retention time of common peaks in batches of finished products

[0124] Peak S1 S2 S3 Mean Relative retention time range ±10% 1(S1) 1.000 1.000 1.000 1.000 / 2 1.169 1.168 1.169 1.169 1.052-1.286 3 1.506 1.506 1.556 1.523 1.370-1.675 4 1.651 1.651 1.658 1.653 1.488-1.818 5 1.730 1.704 1.672 1.702 1.532-1.872 6 1.942 1.946 1.944 1.944 1.750-2.139 7(S2) 1.000 1.000 1.000 1.000 / 8 1.036 1.036 1.036 1.036 0.932-1.140 9 1.123 1.123 1.123 1.123 1.011-1.36 10 1.271 1.271 1.271 1.271 1.144-1.398

[0125] In summary, the standard decoction characteristic spectrum determination method established by HPLC was stability tested in accordance with the "Guidelines for Validation of Analytical Methods (General Chapter 9101)" of Part IV of the 2020 edition of the "Chinese Pharmacopoeia", and met the requirements. According to the characteristic spectrum analysis method proposed above, the characteristic spectra of three batches of finished products were measured, and the results were analyzed. Ten common characteristic peaks were calibrated, and the peak corresponding to the hesperidin reference peak was taken as S1. The relative retention times of peaks 2, 3, 4, 5, and 6 with S1 were calculated. The peak corresponding to the limonin reference peak was taken as S2, and the relative retention times of peaks 8, 9, and 10 with S2 were calculated. The average of the relative retention times of the peaks of the three batches of samples was proposed as the specified values, which were 1.17 (peak 2), 1.53 (peak 3), 1.65 (peak 4), 1.69 (peak 5), 1.94 (peak 6), 1.04 (peak 8), 1.12 (peak 9), and 1.27 (peak 10). Taking into account the errors of multiple factors such as experimental operation, instruments, and reagents, the allowable range of the relative retention time is proposed to be ±10%.

[0126] 4 Extracts

[0127] 4.1 Test methods

[0128] The extraction solvent for Chinese herbal formula granules is pharmaceutical water. According to the "Technical Requirements", ethanol is used as the solvent and the determination is carried out according to the hot infusion method under the alcohol-soluble extract determination method (General Rules 2201 of the 2020 Edition of the Chinese Pharmacopoeia).

[0129] 4.2 Investigation of extractables

[0130] About 2 g of samples from three batches of large-scale production were taken and measured according to the method. The results are shown in Table 17.

[0131] Table 17 - Extraction results

[0132]

[0133] 4.3- Drafting of Extractables Range

[0134] The content of particles was converted from 21 batches of salt orange seed standard decoction extracts, as summarized in Table 18:

[0135] Table 18-21 Summary of extracts from batch 18-21 salt orange seed standard decoction converted into granules

[0136]

[0137] Combined with the paste yield, according to the proposed product specification "each 1g of formula granules is equivalent to 4.5g of decoction pieces", the average extract of 21 batches of standard decoction samples converted into granular extracts is 41.08%, and the measured value converted into granular extracts ranges from 31.59% to 49.56%, with an SD of 5.12; calculated at 70% to 130% of the mean, the allowable range of extracts is 28.76% to 53.41%. Calculated at mean -3SD to mean +3SD, the allowable range of extracts is 25.73% to 56.44%. For this reason, the allowable range of extracts is proposed to be no less than 26.0%. At the same time, combined with the content data of three batches of large-scale production samples, the final limit range of extracts is proposed to be no less than 26.0%.

[0138] 5. Content determination

[0139] 5.1 Basis for selecting the components to be tested

[0140] Citrus reticulata Blanco is the dried mature seed of the Rutaceae plant Citrus reticulata Blanco and its cultivated variants. It is distributed in Fujian, Zhejiang, Guangdong, Guangxi, Jiangxi, Hunan, Guizhou, Yunnan, Sichuan and other places. Salt orange seed is slightly warm, bitter, and slightly oily. It has the effects of promoting qi and relieving pain, softening and dispersing nodules, and dispelling cold. Salt orange seed is a kind of Chinese medicine slice processed by orange seed, and its main material is the dried seeds of orange seed. Orange seed contains limonin, fatty acids, proteins and mineral elements. Orange seed has anti-tumor, anti-inflammatory, analgesic and antibacterial effects. Salt roasting orange seed can enhance its anti-inflammatory and analgesic effects. By consulting the relevant literature "Quality Detection Method and Process of Salt Orange Seed Standard Decoction", it is known that the quality detection method of Salt Orange Seed Standard Decoction uses limonin, nomilin and phlorothalone as reference substances. For this reason, it is of certain reference value to establish the content index of limonin, nomilin and nomilin for the quality detection of Salt Orange Seed Standard Decoction.

[0141] 5.2 Determination of limonin, nomilin and flavonoid content

[0142] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0143] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler SPOLAR C18 S5 (4.6 mm × 250 mm, 5 μm); acetonitrile-water (35:65) was used as the mobile phase, with a flow rate of 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 220 nm, and the theoretical plate number calculated based on the limonin peak should be no less than 5000.

[0144] Preparation of reference solution: Take appropriate amount of limonin, nomilin and phedrine reference substances, weigh accurately, add methanol to make a mixed solution containing 300 μg of limonin, 200 μg of nomilin and 50 μg of phedrine per 1 ml.

[0145] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2g, weigh it accurately, put it in a stoppered conical flask, add 15ml of methanol accurately, stopper it, weigh it, heat it under reflux for 20 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0146] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0147] 5.3 Study on the methodological determination of limonin, nomilin and flavonoids

[0148] 5.3.1 Investigation of extraction methods

[0149] Referring to the method under "5.2" above, samples were extracted in different ways to prepare test solutions, and the test was carried out according to the law. The results showed that the extraction effects of different extraction methods were not much different. In order to facilitate the subsequent experiments, ultrasonic extraction was selected to extract samples, see Table 23 for details.

[0150] Table 23 - Comparison of different extraction methods

[0151]

[0152] 5.3.2 Investigation of extraction time

[0153] Referring to the method under "5.2" above, samples were extracted at different times to prepare test solutions, and the test was performed according to the law. The results showed that ultrasonic extraction for 20 minutes can basically extract the sample completely, so the ultrasonic extraction time of the sample was determined to be 20 minutes, see Table 19 for details.

[0154] Table 19 - Comparison of different extraction times

[0155]

[0156] 5.3.3 Investigation of extraction solvent

[0157] Refer to the method under "5.2" above, extract the samples with different solvents to prepare the test solution, and measure it according to the law. The results show that methanol is the best solvent for extraction, so methanol is selected as the solvent, see Table 20 for details.

[0158] Table 20 - Comparison of different extraction solvents

[0159]

[0160] 5.3.4 Investigation of extraction time

[0161] Referring to the method under "5.2" above, extract samples at different times to prepare test solutions, and perform the test in accordance with the law. The results show that the extraction effect is better when the sample is extracted for 30 minutes, so the extraction time of 30 minutes is selected as the sample extraction time, see Table 21 for details.

[0162] Table 21- Comparison of different extraction times

[0163]

[0164] 5.3.5 Sampling volume investigation

[0165] Referring to the method under "5.2" above, the test solution was prepared with different sampling amounts and the test was carried out according to the law. The results showed that the extraction effect of different sampling amounts was not much different, but 0.2g was extracted more completely, so 0.2g was selected as the sampling amount for preparing the test solution, see Table 22 for details.

[0166] Table 22- Comparison of different sampling volumes

[0167]

[0168] In summary, the main parameters of the test solution preparation method are determined as follows: take an appropriate amount of the product, grind it into powder, take about 0.2 g, accurately weigh it, put it in a stoppered conical flask, accurately add 15 ml of methanol, stopper it, weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0169] 5.4 Validation of analytical method for content determination

[0170] 5.4.1 Specificity inspection

[0171] Take maltodextrin, about 0.2g, and prepare maltodextrin blank control solution according to the method under "5.2" above, and take salt orange seed test solution, maltodextrin blank control solution and reference solution containing 300μg limonin, 200μg nomilin and 50μg flavonoids per 1ml for determination. Fig.15The test showed that the maltodextrin blank had no interference and the method had good specificity.

[0172] 5.4.2 Repeatability test About 0.2 g of salt orange core formula granules (batch number: 211003) were taken, for a total of 6 portions, and the test was carried out according to the test method under the above "5.2". The peak area was measured and the RSD% value of the total content of limonin, nomiline and berberine in the sample was calculated to be 0.85%. The test showed that this method had good repeatability. See Table 23 for details.

[0173] Table 23 - Repeatability test

[0174]

[0175] 5.4.3 Precision test

[0176] Take about 0.2g of salt orange core formula granules (batch number: 211003), and inject 6 times continuously according to the method under the above "5.2", measure the peak area, and calculate the RSD% value of the total content of limonin, nomiline and berberine in the sample to be 1.82%. The test shows that the instrument has good precision. See Table 24 for details.

[0177] Table 24 - Precision test

[0178]

[0179] 5.4.4 Stability test

[0180] Take about 0.2g of salt orange core formula granule sample (batch number: 221001), and according to the test method under the above "5.2", inject the sample at 0h, 2h, 4h, 8h, 12h, and 24h, measure the peak area, and calculate the RSD% value of the total content of limonin, nomiline, and berberine in the sample to be 1.95%. The test shows that the test solution is relatively stable within 24 hours. See Table 25 for details.

[0181] Table 25 - Stability Test

[0182]

[0183] 5.4.5 Sample recovery test

[0184] Accurately weigh about 0.1g of three groups of samples, a total of 9 portions, 3 portions in each group, add 1.20ml, 1.50ml, 1.80ml of a known concentration of limonin reference solution (912.064μg / ml), add 0.20ml, 0.22ml, 0.25ml of a known concentration of nomilin reference solution (552.05μg / ml), and then add 0.20ml, 0.22ml, 0.25ml of a known concentration of flavonoid reference solution (249.60μg / ml). .30ml, 0.38ml, 0.45ml, prepare the test solution according to the method under "5.2", and carry out the determination. The average recovery rate of limonin is 99.20%, and the RSD is 1.73%. See Table 26 for details. The average recovery rate of nomilin is 100.13%, and the RSD is 1.17%. See Table 27 for details. The average recovery rate of berberine is 98.63%, and the RSD is 1.98%. See Table 28 for details.

[0185] Table 26 - Limonin recovery test results

[0186]

[0187] Table 27-Nomilin sample recovery test results

[0188]

[0189] Table 28 - Results of the recovery test of flavonoids

[0190]

[0191] 5.4.6 Durability test

[0192] 5.4.6.1 Experiments with different mobile phase ratios

[0193] Take about 0.2g of salt orange core formula granule sample (batch number: 221103) and test it with acetonitrile-water (36:64), (35:65), and (34:66) according to the test method under "5.2". The calculation results show that the RSD% value of the total content of limonin, nomilin, and chloroquine is 1.03%, which is less than 2.0%. The test shows that this method has good durability to small changes in the mobile phase ratio, see Table 29 for details.

[0194] Table 29 - Comparison of different mobile phase ratios

[0195]

[0196] 5.4.6.2 Tests at different wavelengths

[0197] Take about 0.2g of salt orange core formula granule sample (batch number: 221103), and measure it at wavelengths of 215nm, 220nm, and 225nm according to the test method under "5.2". The calculation results show that the RSD% value of the total content of limonin, nomilin, and chloroquine is 1.94%, which is less than 2.0%. The test shows that this method has good durability against slight changes in wavelength, see Table 30 for details.

[0198] Table 30 - Comparison of different wavelengths

[0199]

[0200] 5.5 Summary

[0201] In summary, the entire analytical method was tested for specificity, peak purity, precision, repeatability, stability, sample recovery and durability, and the results all met the requirements, indicating that the established method can be well used for the determination of limonin, nomiline and berberine.

[0202] 6. Proposed content limit range

[0203] 6.1 Determination of the content of three batches of finished products According to the content analysis method proposed above, the contents of limonin, nomilin and berberine in three batches of finished products were determined. The results are shown in Table 31.

[0204] Table 31-3 Test results of batches of finished products

[0205]

[0206] 6.2 Proposed content limit range

[0207] The content of 21 batches of salt orange seed standard decoction (total content of limonin, nomilin and phedrine) was converted into granule content and summarized as follows:

[0208] Table 32-21 Summary of the content of salt orange seed standard decoction converted into granules

[0209]

[0210] Combined with the paste yield, according to the proposed product specification "each 1g of formula granules is equivalent to 4.5g of decoction pieces", the content of 21 batches of standard decoction samples was converted into granule content, and the average total content of limonin, nomilin, and flavonoids was 12.49mg / g, and the measured content was converted into granule content in the range of 8.427mg / g to 16.979mg / g, with an SD of 2.11; calculated at 70% to 130% of the mean, the allowable range of the total content of limonin, nomilin, and flavonoids is 8.74mg / g to 16.24mg / g. Calculated by mean -3SD to mean +3SD, the allowable range of the total content of limonin, nomilin, and flavonoids is 6.16mg / g to 18.82mg / g. Combining the two calculation methods, the upper limit of the allowable range is taken, so the allowable range of the total content of limonin, nomilin, and flavonoids is proposed to be 8.7mg / g to 18.8mg / g.

[0211] After comprehensive comparison and combined with the content data of three batches of large-scale production samples, the final total content range of limonin, nomiline and berberine was proposed to be 8.7 mg / g to 18.8 mg / g.

[0212] 7 Specifications are determined based on the amount of raw materials added in the preparation method and the amount produced through process research, that is, every 1g of formula granules is equivalent to 4.5g of medicinal pieces.

[0213] 8 Storage is formulated according to the basic storage requirements of the variety.

[0214] The quality detection method of salt orange core formula granules provided by the present invention studies the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and total content determination of limonin, nomilin and flavonoids of salt orange core formula granules, and evaluates the quality of salt orange core formula granules through multi-faceted measurements, laying a solid foundation for the stable quality of the product, and can establish a feasible quality standard for salt orange core formula granules, and realize effective control of the quality of salt orange core formula granules. Moreover, liquid phase analysis can be performed using the chromatographic conditions of the present application to obtain a characteristic spectrum with better separation and clearer characteristics. The method has good stability, high precision and good reproducibility. Salt orange core decoction pieces are decocted to obtain salt orange core decoction pieces, and the total content of limonin, nomilin and flavonoids is 12.49 mg / g on average. According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", the total content of limonin, nomilin and flavonoids is set to be 8.7-18.8 mg / g.

[0215] Those skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention (including claims) is limited to these examples; any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the quality of salt orange seed formula particles, characterized in that: The following detection methods are included: Through the properties of salt orange seed formula granules, dry extract yield, thin layer identification, characteristic spectrum, extract and total content of limonin, nomilin and flavone, the content standard of the formula granules is limited to 8.7-18.8 mg of limonin, nomilin and flavone per 1g. Among them, thin layer identification is carried out by thin layer chromatography; characteristic spectrum and total content of limonin, nomilin and flavone are determined by liquid chromatography; extract is determined by hot immersion method; The characteristic spectrum is determined by liquid chromatography, including: performing liquid chromatography analysis, using a solution made from a tangerine seed control medicinal material as a control medicinal material reference solution b, using a solution made from a limonin and hesperidin reference substance as a reference substance reference solution b, using a solution made from a salt tangerine seed formula granule sample as a test solution b, accurately aspirating the control medicinal material reference solution b, the reference substance reference solution b and the test solution b, respectively, injecting them into a liquid chromatograph, and determining, to obtain; wherein the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 µm; a mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution according to the provisions of Table a; Table a Gradient elution program Flow rate: 1.2 mL / min; column temperature: 30 °C; injection volume: 10 μL; detection wavelength: 220 nm; The liquid chromatography method for determining the total content of limonin, nomilin and phloxetine includes: performing liquid chromatography analysis, using a solution made from limonin, nomilin and phloxetine reference substances as the reference solution c, and using a solution made from a salt orange seed formula granule sample as the test solution c, accurately aspirating the reference solution c and the test solution c, respectively, injecting them into the liquid chromatograph, and determining to obtain the content; wherein, the chromatographic conditions used are: chromatographic column: octadecylsilane bonded silica gel as the filler, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5µm; mobile phase: acetonitrile-water with a volume ratio of 35:65 as the mobile phase; flow rate: 1.0 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 220 nm.

2. The quality detection method of salt orange seed formula granules according to claim 1, characterized in that: The preparation method of the salt orange seed formula granules is as follows: take 4500g of salt orange seed pieces, add water to boil, filter, concentrate the filtrate into a clear paste, add granule auxiliary materials, dry or dry and crush, then add granule auxiliary materials, mix well, granulate, and make 1000g.

3. The quality detection method of salt orange seed formula granules according to claim 1, characterized in that, The thin layer chromatography method comprises the following steps: (1) Preparation of test solution a: Take 1.0 g of salt orange seed granules, grind them into powder, add 20 mL of petroleum ether, ultrasonically treat for 30 min, filter, discard the filtrate, add 20 mL of dichloromethane to the residue, ultrasonically treat for 20 min, filter, evaporate the filtrate, and dissolve the residue in 1 mL of methanol to prepare test solution a; (2) Preparation of control medicinal material solution a: Take 1.0 g of orange seed control medicinal material, grind it into powder, add 20 mL of petroleum ether, ultrasonically treat it for 30 min, filter it, discard the filtrate, add 20 mL of dichloromethane to the residue, ultrasonically treat it for 20 min, filter it, evaporate the filtrate to dryness, add 1 mL of methanol to the residue to dissolve it, and prepare control medicinal material solution a; (3) Thin layer chromatography analysis: Thin layer chromatography conditions are as follows: Thin layer plate: silica gel G thin layer plate; sample volume: 4ul of test solution a, 8ul of control drug solution a; developing solvent: dichloromethane-ethyl acetate solution with a volume ratio of 4:1 as the developing solvent; Color developer: dimethylbenzylidene rhodanine-sulfuric acid-ethanol solution with a mass percentage of 1:2:97, heated at 105°C, and inspected under ultraviolet light at 365nm.

4. The quality detection method of salt orange seed formula granules as claimed in claim 3, characterized in that, The petroleum ether is 60-90°C petroleum ether.

5. The quality detection method of salt orange seed formula granules according to claim 1, characterized in that, The hot soaking method uses ethanol as solvent, and the hot soaking method under the alcohol-soluble extract determination method is used to determine the extract range.

6. The quality detection method of salt orange seed formula granules according to claim 1, characterized in that: The method of measuring the characteristic spectrum by liquid chromatography also includes the following steps: (1) Preparation of reference medicinal material solution b: Take 0.5 g of orange seed reference medicinal material, add 15 mL of methanol, reflux for 50 min, cool, filter, and take the filtrate as the reference medicinal material solution b; (2) Preparation of reference substance solution b: Take appropriate amount of limonin and hesperidin reference substances, weigh accurately, and add methanol to prepare a mixed solution containing 500 µg limonin and 10 µg hesperidin per 1 ml, which is used as reference substance solution b; (3) Preparation of test solution b: Take an appropriate amount of salt orange seed granules, grind them into powder, take about 0.4 g, place in a stoppered conical flask, add 15 mL of methanol, heat and reflux for 30 min, cool, shake well, filter, and take the filtrate as the test solution b.

7. The quality detection method of salt orange seed formula granules according to claim 1, characterized in that: The method of using liquid chromatography to determine the total content of limonin, nomilin and chloroquine also includes the following steps: (1) Preparation of reference solution: Take appropriate amount of limonin, nomilin and phloxetine, weigh accurately, add methanol to prepare a mixed solution containing 300 μg limonin, 200 μg nomilin and 50 μg phloxetine per 1 ml, as reference solution c; (2) Preparation of test solution: Take an appropriate amount of salt orange seed granules, grind them into powder, take about 0.2 g, weigh accurately, place in a stoppered conical flask, accurately add 15 ml of methanol, stopper, weigh, heat under reflux for 20 min, cool, weigh again, make up the loss with methanol, shake well, filter, and take the filtrate as the test solution c.

Images