A method for quality control of a sample of a coleus

Abstract

The application discloses a quality control method of a justicia sample, which comprises construction of a characteristic map of the justicia sample and content determination of the justicia sample, wherein the characteristic map comprises at least seven characteristic peaks, reference peaks in the characteristic peaks correspond to justicidin A, justicidin B and 6'-hydroxy justicidin C reference substances respectively, and the content of justicidin A and justicidin B in each gram of the sample is in the range of 0.3 mg to 1.50 mg and 0.10 mg to 0.80 mg. The optimized detection method is used for comprehensively monitoring the medicinal materials, decoction pieces, preparation intermediates and formula granules of the justicia, and the method is simple in operation, good in reproducibility, accurate and reliable, time-saving, and provides a new analysis method for internal quality control of the justicia.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for quality control of a drug, in particular to a method for quality control of Justicia procumbens L. BACKGROUND

[0002] Justicia procumbens L. is the dried whole plant of Justicia procumbens L. (State Pharmacopoeia Commission of P. R. C. Chinese Pharmacopoeia (Part I) [S]. 1977: 279). Also known as Jueqing (Wu Pu Ben Cao), Xiangsu (Bie Lu), Dragonfly Chapter, Fly Wing (Gaimu Diyi), Red Eye Old Mother Grass (Xin Xi Ben Cao), Red Eye (Pin Hui Jing Yao) and so on. Bitter, salty, pungent, cold, and belongs to the lung, liver and bladder. Clearing heat and resolving toxins, reducing dampness and eliminating accumulation, promoting blood circulation and relieving pain. It is used to treat cold, cough, sore throat, red and swollen eyes, malnutrition, damp-heat diarrhea, malaria, jaundice, edema, turbid urine, muscle and bone pain, sprains, carbuncles, sores, eczema, etc. (Chinese Academy of Sciences, Chinese Plant Editorial Committee. Chinese Plant Flora [M]. Science Press, 2013.) Modern research shows that the main components of Justicia procumbens L. are Justicin A, 6'-hydroxy Justicin B, Justicin B, Justicidin, β-daucosterol, ruscogenin, stigmasterol, and new Justicin A (Lü Jinpeng, Jin Hong, Zhao Menghui, et al. Study on the chemical constituents of Justicia procumbens L. [J]. Military Medicine, 2019, 43(3): 207-212.) Modern research shows that it also has anti-tumor, anti-virus, anti-platelet aggregation effects, and can be used for pediatric malnutrition, intractable diarrhea, acute urinary tract infection in women, etc. (Liu Guorui, Wu Jun, Yang Meihua, et al. Research progress of medicinal plant Justicia procumbens L. [J]. Northwest Pharmaceutical Journal, 2008, (1): 55-56.) Studies have shown that Justicia procumbens L. contains rich lignans, and Justicidin has anti-platelet aggregation and anti-tumor activity (Peng Jingling. Mechanism of anti-platelet aggregation of effective substances in Justicia procumbens L. [D]. Hubei University of Chinese Medicine, 2019), which is a specific index component of Justicia procumbens L.

[0003] The quality research of the present basil mainly includes the following aspects: Gan Jiaoye et al. studied the quality control method of basil medicinal materials, including water content test, total ash and acid insoluble ash test, thin layer identification, microscopic identification and content determination of effective ingredient labiatae fat B (Gan Jiaoye, Yang Yanfang, Hong Zongchao, et al. Study on Quality Standard of Basil Medicinal Materials [J]. Asia-Pacific Traditional Medicine, 2017, 13(23): 30-33.). Xu Jingjing et al. established HPLC chromatogram of basil medicinal materials from 17 different producing areas, and determined the contents of 6'-hydroxy-labiatae fat B and labiatae fat B, two common ingredients, for analyzing and detecting the difference of basil medicinal material ingredients (Xu Jingjing, Li Qiji, Yang Yan, et al. Study on the Difference of HPLC Components of Basil Medicinal Materials from Different Producing Areas [J]. Chinese Herbal Medicines, 2017, 40(08): 1888-1891.). Yang Yanfang et al. determined the contents of 6'-hydroxy-labiatae fat B, new labiatae fat B and Taiwan E in basil medicinal materials by HPLC method (Yang Yanfang, Chen Qingjie, Li Tingting, et al. Simultaneous Determination of 6'-Hydroxy-labiatae Fat B, New Labiatae Fat B and Taiwan E in Basil Medicinal Materials by RP-HPLC [J]. Journal of Pharmaceutical Analysis, 2013, 33(1): 120-123.).

[0004] But the above-mentioned method has a long detection and analysis period, and most of the quantitative control products are laboratory self-made products, which are not suitable as the content determination index of medicinal materials, decoction pieces and formula granules, and the universality of the standard evaluation method is poor.

[0005] In addition, the thin layer identification of basil and the lower limit of labiatae fat B content are specified in Hubei Province Chinese Herbal Medicine Processing Standard (Hubei Provincial Drug Administration. Hubei Province Chinese Herbal Medicine Processing Standard [M]. 2019). It is difficult to comprehensively control the internal quality of basil by studying only one index component, and it is impossible to detect the quality of basil and its preparations as a whole. SUMMARY

[0006] The purpose of the present application is to overcome the defects that the quality control method of basil products in the prior art cannot comprehensively monitor the quality or is time-consuming and laborious, and to provide a quality control method for basil samples with good reproducibility, stability, reliable recovery rate, low detection cost and high detection efficiency.

[0007] The technical scheme of the present application is a quality control method for basil samples, which comprises the construction of a characteristic map of the basil sample and the content determination of the basil sample, the characteristic map comprising at least 7 characteristic peaks, the reference peaks in the characteristic peaks corresponding to labiatae fat A, labiatae fat B and 6'-hydroxy-labiatae fat C control respectively, and the contents of labiatae fat A and labiatae fat B in each gram of sample being in the range of 0.3 mg-1.50 mg and 0.10 mg-0.80 mg.

[0008] Further, the construction method of the sample characteristic spectrum of Plumbago zeylanica L, comprises the following steps:

[0009] (1) Select Plumbago zeylanica L samples and prepare test sample solutions; prepare reference substance solutions of Plumbago zeylanica L control medicinal materials with Plumbago zeylanica L as the control medicinal material; prepare reference substance solutions of control substances with Plumbago zeylanica L ester A, Plumbago zeylanica L ester B and 6'-hydroxy Plumbago zeylanica L ester C as the control substances;

[0010] (2) Take the reference substance solutions and the test sample solutions respectively for ultra-high performance liquid chromatography analysis to construct the characteristic spectrum of the sample to be measured.

[0011] Further, take 0.2g-1.0g of Plumbago zeylanica L formula granules, add an organic solvent for extraction, and take the subsequent filtrate to obtain the test sample solution; take 0.2g-1.0g of Plumbago zeylanica L control medicinal materials, add an organic solvent for extraction, and take the subsequent filtrate to obtain the reference substance solution of the control medicinal materials.

[0012] Still further, the extraction agent for preparing the reference substance solution or / and the test sample solution is water or a methanol solution with a volume concentration of 50%-100%.

[0013] Still further, take 0.2g-1.0g of Plumbago zeylanica L formula granules, grind them finely, add 15-50ml of a methanol aqueous solution with a volume concentration of 50%-100% for ultrasonic extraction, and take the subsequent filtrate to obtain the test sample solution; take 0.2g-1.0g of Plumbago zeylanica L control medicinal materials, add 30-100ml of a methanol aqueous solution with a volume concentration of 50%-100% for ultrasonic extraction, and take the subsequent filtrate as the reference substance solution of the control medicinal materials; additionally take appropriate amounts of Plumbago zeylanica L ester A, Plumbago zeylanica L ester B and 6'-hydroxy Plumbago zeylanica L ester C control substances, dissolve them in a methanol aqueous solution as the reference substance solution of the control substances; the ultrasonic extraction time is 15-60 minutes.

[0014] Further, the concentration of the Plumbago zeylanica L ester A, Plumbago zeylanica L ester B and 6'-hydroxy Plumbago zeylanica L ester C control substance reference substance solutions in step (1) is 5-50μg / ml.

[0015] Still further, in the preparation of the reference substance solution, the amount of extraction agent used per gram of Plumbago zeylanica L ester A, Plumbago zeylanica L ester B and 6'-hydroxy Plumbago zeylanica L ester C is 30ml-100ml, and the extraction time is 15-60 minutes.

[0016] Further, in step (2), 0.5μl-2μl of the reference substance solution and the test sample solution are taken respectively for determination.

[0017] Further, the conditions of the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is filled with octadecylsilane-bonded silica gel; acetonitrile is used as the mobile phase A, and 0.05%-0.2% phosphoric acid solution is used as the mobile phase B, and gradient elution is adopted: 0-6 min, 27→31% A, 6-10 min, 31→38% A, 10-15 min, 38→65% A, 15-18 min, 65→80% A, 18-21 min, 80% A, 21-22 min, 80→27% A.

[0018] Further, the detection wavelength is 254-280 nm; the flow rate is 0.25 ml-0.35 ml per minute; and the column temperature is 20°C-30°C; and an ultraviolet detector is used.

[0019] Further, the ultra-high performance liquid chromatography characteristic chromatogram of the justicia sample has 7 characteristic peaks; and the 7 characteristic peaks correspond to the retention time of the characteristic peaks of the reference substance chromatogram of the control medicinal material; wherein peak 5, peak 6 and peak 7 correspond to the retention time of the reference substance peaks of justicidin B control, justicidin A control and 6'-hydroxy justicidin C control respectively. The peak corresponding to the reference substance peak of justicidin B control is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified value is: 0.38 (peak 1), 0.40 (peak 2), 0.61 (peak 3), 0.72 (peak 4).

[0020] The content determination method of the justicia sample comprises the following steps:

[0021] (1) Selecting a justicia sample and preparing a test sample solution; and preparing a control reference solution by using justicidin A and justicidin B as the control reference;

[0022] (2) Performing ultra-high performance liquid chromatography analysis on the control reference solution and the test sample solution respectively to construct a content determination method for the sample to be tested, and the content indicators are justicidin A and justicidin B.

[0023] Further, in step (1), 0.2 g-1.0 g of justicia formula granules is finely ground, 50%-100% methanol is added for extraction, and the filtrate is taken to obtain the test sample solution; and an appropriate amount of justicidin A and justicidin B control is dissolved in a methanol aqueous solution to serve as the control reference solution.

[0024] Further, 0.2 g-1.0 g of justicia formula granules is extracted with an organic solvent, and the filtrate is taken to obtain the test sample solution. The extractant for preparing the test sample solution is a methanol solution with a volume concentration of 70%-100%.

[0025] Further, the amount of extraction solvent used for preparing the test sample solution is 30-100 ml per gram of pectolinarigenin A and pectolinarigenin B, and the extraction time is 15-60 minutes.

[0026] Further, the concentration of the reference solution of pectolinarigenin A and pectolinarigenin B in step (1) is 5-50 μg / ml.

[0027] Further, 0.5-2 μl of the reference solution and the test sample solution are taken respectively in step (2).

[0028] Further, the conditions of the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is filled with octadecylsilane-bonded silica gel; the mobile phase is acetonitrile-0.05%-0.2% phosphoric acid solution (45:55); the detection wavelength is 190-400 nm; the flow rate is 0.25-0.35 ml per minute; the column temperature is 25-35 °C; and an ultraviolet detector is used.

[0029] Further, the sample of Plumbago zeylanica is Plumbago zeylanica medicinal material, Plumbago zeylanica decoction pieces, Plumbago zeylanica preparation intermediate or Plumbago zeylanica formula granules.

[0030] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0031] (1) The quality control method of the Plumbago zeylanica sample is simple, fast and effective, and controls the quality from the aspects of characteristic spectrum and content determination, thereby providing stronger guarantee for the industrialization of Plumbago zeylanica sample, and being suitable for Plumbago zeylanica medicinal material, Plumbago zeylanica decoction pieces, Plumbago zeylanica preparation intermediate or Plumbago zeylanica preparation finished product;

[0032] (2) The method is simple to operate, has good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency, and provides important guarantee for the standardization of the production of Plumbago zeylanica sample. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Fig. 4 is a characteristic spectrum of Plumbago zeylanica formula granules, wherein peak 5 (S) is pectolinarigenin B; peak 6 is pectolinarigenin A; and peak 7 is 6'-hydroxyl pectolinarigenin C.

[0034] Figure 2 Fig. 5 is a characteristic spectrum of Plumbago zeylanica intermediate, wherein peak 5 (S) is pectolinarigenin B; peak 6 is pectolinarigenin A; and peak 7 is 6'-hydroxyl pectolinarigenin C.

[0035] Figure 3 Fig. 6 is a characteristic spectrum of Plumbago zeylanica medicinal material / decoction pieces, wherein peak 5 (S) is pectolinarigenin B; peak 6 is pectolinarigenin A; and peak 7 is 6'-hydroxyl pectolinarigenin C.

[0036] Figure 4 Fig. 7 is a chromatogram of a reference substance, a reference medicinal material and Plumbago zeylanica formula granules.

[0037] Figure 5 Full wavelength scan of rosmaridin B;

[0038] Figure 6 Full wavelength scan of rosmaridin A;

[0039] Figure 7 UPLC chromatogram of rosmarinus officinalis formula granules at different detection wavelengths;

[0040] Figure 8 Characteristic UPLC chromatogram of different extraction solvents;

[0041] Figure 9 Characteristic UPLC chromatogram of different extraction methods;

[0042] Figure 10 Characteristic UPLC chromatogram of different extraction times;

[0043] Figure 11 Characteristic UPLC chromatogram of different extraction volumes;

[0044] Figure 12 Specificity test of rosmarinus officinalis formula granules characteristic chromatogram;

[0045] Figure 13 Integrity test of rosmarinus officinalis formula granules;

[0046] Figure 14 Chromatographic column investigation of rosmarinus officinalis formula granules characteristic chromatogram;

[0047] Figure 15 Column temperature investigation of rosmarinus officinalis formula granules characteristic chromatogram;

[0048] Figure 16 Flow rate investigation of rosmarinus officinalis formula granules characteristic chromatogram;

[0049] Figure 17 Content UPLC chromatogram of different extraction solvents;

[0050] Figure 18 Content UPLC chromatogram of different extraction methods;

[0051] Figure 19 Content UPLC chromatogram of different extraction times;

[0052] Figure 20 Content UPLC chromatogram of different extraction volumes;

[0053] Figure 21 Linear relationship diagram of rosmaridin B reference substance;

[0054] Figure 22Linear relationship chart for spilantholide A reference substance;

[0055] Figure 23 Specificity test of content of spilantholide granules;

[0056] Figure 24 Overall test of content of spilantholide granules;

[0057] Figure 25 Column investigation of content of spilantholide granules;

[0058] Figure 26 Column temperature investigation of specific content of spilantholide granules;

[0059] Figure 27 Flow rate investigation of content of spilantholide granules. DETAILED DESCRIPTION

[0060] The technical solutions of the present application are further described below in combination with the drawings.

[0061] Example 1 Quality control determination of spilantholide granules

[0062] Take 3 batches of spilantholide granules and 9 batches of spilanthes medicinal materials as spilanthes formula samples, and perform quality control determination according to the following steps:

[0063] 1. Construction method of characteristic spectrum

[0064] (1) Select spilanthes samples and prepare test sample solutions; take spilanthes as the control medicinal material, prepare a reference solution of the control medicinal material; take spilantholide A, spilantholide B and 6'-hydroxyspilantholide C as reference substances, and prepare reference solutions of the control substances respectively.

[0065] Preparation of reference solution: take 0.5 g of spilanthes control medicinal material, place it in a conical flask with a plug, add 25 ml of 70% methanol, tightly plug, ultrasonically treat (power 600 W, frequency 40 kHZ) for 30 minutes, cool, shake well, filter, take the filtrate as the reference solution of the control medicinal material.

[0066] Preparation of reference solution of control substance: take appropriate amounts of spilantholide A, spilantholide B and 6'-hydroxyspilantholide C reference substances, accurately weigh, add methanol to prepare a mixed solution containing 10 μg per 1 ml, and obtain it.

[0067] Preparation of test sample solution of spilanthes formula granules: take about 0.5 g of spilanthes formula granules, accurately weigh, place it in a conical flask with a plug, accurately add 25 ml of methanol, tightly plug, weigh, ultrasonically treat (power 600 W, frequency 40 kHZ) for 45 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and obtain it.

[0068] Preparation of the test solution of the sample of Justicia procumbens L. : take Justicia procumbens L. powder 0.5 g, accurately weigh, put into a conical flask with a plug, accurately add 70% methanol 25 ml, tightly plug, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and obtain.

[0069] (2) The test solution and the reference solution prepared in step (1) are subjected to ultra-high performance liquid chromatography analysis respectively to obtain the corresponding ultra-high performance liquid chromatogram; 1 μl of the reference solution and the test solution is accurately taken respectively and injected into the liquid chromatograph for determination, and thus the characteristic chromatogram of Justicia procumbens L. is obtained.

[0070] The chromatographic condition parameters are as follows:

[0071] The octadecylsilane-bonded silica gel is used as the filler (the column length is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm); the acetonitrile is used as the mobile phase A, and the 0.1% phosphoric acid solution is used as the mobile phase B, which is subjected to gradient elution according to the following table; the flow rate is 0.30 ml per minute; the column temperature is 25 °C; and the detection wavelength is 254 nm.

[0072]

[0073] The Justicia procumbens L. formula granules can be replaced by other Justicia procumbens L. samples, such as Justicia procumbens L. medicinal materials, Justicia procumbens L. decoction pieces, Justicia procumbens L. preparation intermediates, or Justicia procumbens L. preparation finished products. The Justicia procumbens L. intermediate is selected as the Justicia procumbens L. sample, step (1) is repeated, and the characteristic chromatogram of the Justicia procumbens L. intermediate is constructed as shown in Figure 2 The Justicia procumbens L. medicinal material / decoction piece is selected as the Justicia procumbens L. sample, step (1) is repeated, and the characteristic chromatogram of the Justicia procumbens L. medicinal material / decoction piece is constructed as shown in Figure 3

[0074] As shown in Figure 1 and 4 , the characteristic chromatogram of the Justicia procumbens L. formula granules, seven characteristic peaks are present in the test solution chromatogram, which correspond to the retention times of the seven characteristic peaks in the reference chromatogram of the control medicinal material, wherein the peak 5, the peak 6, and the peak 7 correspond to the retention times of the Justicia procumbens L. ester B control, the Justicia procumbens L. ester A control, and the 6'-hydroxy Justicia procumbens L. ester C control, respectively. The peak corresponding to the Justicia procumbens L. ester B control is the S peak, and the relative retention times of the other characteristic peaks to the S peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values are: 0.38 (peak 1), 0.40 (peak 2), 0.61 (peak 3), and 0.72 (peak 4).

[0075] The determination results of the test solution are shown in the following table.

[0076] Table 1 Determination results of the Justicia procumbens L. formula granule sample (relative retention time)​

[0077]

[0078] Table 2 Determination results of 9 batches of samples of Ocimum tenuiflorum L. medicinal materials (relative retention time)

[0079]

[0080] (3) Comparing the ultra-high performance liquid chromatogram obtained in step (2) with the characteristic chromatogram in step (1), qualitative analysis of the sample is carried out;

[0081] 2. Content determination of ocimarensin A and ocimarensin B

[0082] (1) Selecting Ocimum tenuiflorum L. samples and preparing test sample solutions; taking ocimarensin A and ocimarensin B as reference substances, reference substance solutions are prepared;

[0083] Preparation of reference substance solutions: taking appropriate amounts of ocimarensin A and ocimarensin B reference substances, accurately weighing, adding methanol to prepare a mixed solution containing 10 μg per 1 ml.

[0084] Preparation of test sample solutions of Ocimum tenuiflorum L. formula granules: taking about 0.5 g of Ocimum tenuiflorum L. formula granules, accurately weighing, placing in a conical flask with a plug, accurately adding 25 ml of methanol, tightly sealing, weighing, ultrasonic treatment (power 600 W, frequency 40 kHZ) for 45 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking evenly, filtering, and taking the filtrate, which is obtained.

[0085] Preparation of test sample solutions of Ocimum tenuiflorum L. medicinal materials / pieces: taking about 0.5 g of the powder (passed through a No. 3 sieve) of the product, accurately weighing, placing in a conical flask with a plug, accurately adding 25 ml of 70% methanol, tightly sealing, weighing, ultrasonic treatment (power 600 W, frequency 40 kHZ) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking evenly, filtering, and taking the filtrate, which is obtained.

[0086] (2) The test sample solution and the reference substance solution prepared in step (1) are subjected to ultra-high performance liquid chromatography analysis to obtain corresponding ultra-high performance liquid chromatograms; 1 μl of the reference substance solution and the test sample solution are accurately taken and injected into the liquid chromatograph for determination, and then obtained.

[0087] Among them, the chromatographic condition parameters are as follows:

[0088] Octadecylsilane-bonded silica gel is used as the filler (the column length is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm); acetonitrile-0.1% phosphoric acid solution (45:55) is used as the mobile phase for isocratic elution; the flow rate is 0.30 ml per minute; the column temperature is 30℃; and the detection wavelength is 262 nm.

[0089] (3) Taking prenylactonidine A and prenylactonidine B as the content determination indicators, quantitative analysis of the sample to be tested was performed by the content determination method obtained in step (2).

[0090] The contents of prenylactonidine A and prenylactonidine B in the sample were determined, and the experimental results are shown in Tables 3 and 4.

[0091] Table 3 Determination of the content of prenylactonidine A in the jiaocheng formula granules

[0092]

[0093] Table 4 Determination of the content of prenylactonidine A in the jiaocheng medicinal materials

[0094]

[0095] From the above test results, it can be seen that, from the qualitative analysis, the characteristic chromatograms of the 3 batches of jiaocheng formula granules and the 9 batches of jiaocheng medicinal materials all meet the requirements of the characteristic chromatogram in step (2). From the quantitative analysis, the contents of prenylactonidine A and prenylactonidine B in the 3 batches of jiaocheng formula granules and the 9 batches of jiaocheng medicinal materials are stable, and the content of jiaocheng is more than 0.051%, which ensures the effectiveness of the jiaocheng formula granules and the medicinal materials.

[0096] Example 2 Establishment of the characteristic chromatogram method of the jiaocheng formula granules and its methodological study

[0097] This example is used to confirm the optimal scheme of the establishment of the characteristic chromatogram in the quality control method of the jiaocheng sample, which specifically includes the comparison of the schemes of the chromatographic conditions, wavelength selection, and preparation of the test solution, and the selection of the optimal chromatographic conditions and the test solution preparation scheme; and the specificity, integrity, precision, intermediate precision, stability, repeatability, and durability of the optimal scheme are investigated.

[0098] 1. Instruments and reagents

[0099] Agilent high performance liquid chromatograph (1290 Infinity, Agilent Corporation); Waters high performance liquid chromatograph (Waters H-Class, Waters Corporation); one-hundredth scale balance (AP224W, Mettler-Toledo Corporation); millionth scale balance (XBR36, Mettler-Toledo Corporation); numerical control ultrasonic cleaner (2700TH, Shanghai Anpu Experimental Technology Co., Ltd.); electric heating constant temperature water bath (J-HH-6A, Shanghai Shengwei Electronic Technology Co., Ltd.); ultrapure water system (Comfort1, Germany Sartorius Corporation); methanol (AR, Tianjin Damao Chemical Reagent Co., Ltd.); phosphoric acid (HPLC, Shanghai Maikelin Biochemical Technology Co., Ltd.); acetonitrile (HPLC, CNW Corporation); water is ultrapure water; and other reagents are all analytical pure.

[0100] Jadein A reference substance, Jadein B reference substance, 6'-hydroxyjadein C reference substance were purchased from Chengdu Pusai Biotechnology Co., Ltd., batch numbers were PS013032, PS013033, PS013034 respectively, and the purity was 98%.

[0101] Jadeite reference drug was purchased from China Institute for Drug Control, batch number was 121176-201003.

[0102] Jadeite formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0103] 2. Determination of detection wavelength

[0104] Take the product, grind finely, take about 0.5g, accurately weigh, put in a conical flask with a plug, accurately add 25ml of methanol, seal tightly, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 45 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate as the test solution, record the chromatogram at different wavelengths, as shown in Figure 7 .

[0105] The results showed that the chromatographic information detected at 254nm was more abundant, and the detection wavelength of 254nm was finally selected as the detection wavelength.

[0106] 3. Determination of chromatographic conditions

[0107] Octadecylsilane-bonded silica gel was used as the filler (column length 100mm, inner diameter 2.1mm, particle size 1.8μm); 0.1% phosphoric acid was used as the mobile phase A, acetonitrile as the mobile phase B, gradient elution was carried out according to the following table; flow rate was 0.3ml / min; column temperature was 25℃; detection wavelength was 254nm.

[0108]

[0109] 4. Preparation of test solution

[0110] 4.1 Investigation of different extraction solvents

[0111] This experiment investigated the influence of different extraction solvents on the characteristic chromatogram of jadeite formula granules. Water, 30% methanol, 50% methanol, 70% methanol and methanol were selected as extraction solvents, and the peak area / sample weight of 7 chromatographic peaks and chromatogram were used to compare the influence of different extraction solvents on the characteristic chromatogram of jadeite formula granules.

[0112] Take the product (batch number: 21070109) appropriate amount, grind, take about 0.5g, parallel 5, precision weighing, in the conical flask with plug, precision add water, 30% methanol, 50% methanol, 70% methanol, methanol 25ml, tight plug, weigh, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes, cool, weigh again, with the corresponding solvent to make up for the loss of weight, shake, filter, take the filtrate, that is. According to the chromatographic conditions in the text, the results are shown in table 5, Figure 8 .

[0113] Table 5 comparison of extraction efficiency of different extraction solvents (peak area / weight)

[0114]

[0115] The results show that the extraction efficiency of 70% methanol and methanol as extraction solvent is close, combined with the content determination investigation results, finally choose methanol as extraction solvent.

[0116] 4.2 Investigation of different extraction methods

[0117] This experiment investigates the influence of different extraction methods on the characteristic chromatogram of the formula granules of the plant. Ultrasonic treatment, heating reflux and shaking extraction are selected as the extraction methods. The peak area / weight of 7 chromatographic peaks and the chromatogram are compared to investigate the influence of different extraction methods on the characteristic chromatogram of the formula granules of the plant.

[0118] Take the product (batch number: 21070109) appropriate amount, grind, take about 0.5g, parallel 3, precision weighing, in the conical flask with plug, precision add methanol 25ml, tight plug, weigh, ultrasonic treatment (power 600W, frequency 40kHz), shaking extraction, heating reflux 30 minutes, cool, weigh again, with methanol to make up for the loss of weight, shake, filter, take the filtrate, that is. According to the chromatographic conditions in the text, the results are shown in table 6, Figure 9 .

[0119] Table 6 comparison of extraction efficiency of different extraction methods (peak area / weight)

[0120]

[0121] The results show that the extraction efficiency of ultrasonic and heating reflux is close. In order to facilitate experimental operation, the extraction method is finally determined as ultrasonic treatment.

[0122] 4.2 Investigation of different extraction times

[0123] The experiment respectively investigates the influence of four different extraction time, 15 minutes, 30 minutes, 45 minutes and 60 minutes, on the characteristic chromatogram of the Plumbago zeylanica Formula Granules, and compares the influence of different extraction time on the characteristic chromatogram of the Plumbago zeylanica Formula Granules through the peak area / sample weight of 7 chromatographic peaks and chromatograms.

[0124] Take the product (batch number: 21070109) in an appropriate amount, grind finely, take about 0.5g, parallel 4 times, accurately weigh, place in a conical flask with a plug, accurately add 25ml of methanol, tightly plug, weigh, ultrasonic treatment (power 600W, frequency 40kHZ) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and get it. Sample by chromatographic conditions under the text, the results are shown in Table 7, Figure 10 .

[0125] Table 7 Comparison of extraction efficiency of different extraction time (peak area / sample weight)

[0126]

[0127] The results show that the extraction efficiency of different extraction time is not much different, combined with the determination of content, the extraction time is finally determined as 45 minutes.

[0128] 4.3 Investigation of different extraction volumes

[0129] The experiment respectively investigates the influence of three different extraction solvent volumes, 15ml, 25ml and 50ml, on the characteristic chromatogram of the Plumbago zeylanica Formula Granules, and compares the influence of different extraction volumes on the characteristic chromatogram of the Plumbago zeylanica Formula Granules through the peak area / sample weight*extraction volume / 25 of 7 chromatographic peaks and chromatograms.

[0130] Take the product (batch number: 21070109) in an appropriate amount, grind finely, take about 0.5g, parallel 3 times, accurately weigh, place in a conical flask with a plug, accurately add 15ml, 25ml and 50ml of methanol respectively, tightly plug, weigh, ultrasonic treatment (power 600W, frequency 40kHZ) for 45 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and get it. Sample by chromatographic conditions under the text, the results are shown in Table 8, Figure 11 .

[0131] Table 8 Comparison of extraction efficiency of different extraction volumes (peak area / sample weight*extraction volume / 25)

[0132]

[0133] The results show that the extraction efficiency of 25ml and 50ml is equivalent, considering the saving of solvent, the extraction volume is finally determined as 25ml.

[0134] 4.4 Determination of the preparation method of test solution

[0135] According to the above research results, the preparation method of test solution of Plumbago zeylanica Formula Granules characteristic map is determined as follows:

[0136] Take the product, grind finely, take about 0.5 g, accurately weigh, place in a conical bottle with a plug, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 45 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and obtain.

[0137] 5. Methodology study of characteristic map

[0138] 5.1. Study of specificity

[0139] The auxiliary materials added in Plumbago zeylanica Formula Granules are malt dextrin, silicon dioxide and magnesium stearate. This experiment investigates the influence of negative samples without Plumbago zeylanica on Plumbago zeylanica Formula Granules. The negative sample solution is prepared according to the test solution preparation method, 1 μl of sample is injected according to the chromatographic conditions, and the results are shown below Figure 12 .

[0140] The results show that the solvent and auxiliary materials do not interfere with the determination of the characteristic map of Plumbago zeylanica Formula Granules.

[0141] 5.2. Study of integrity

[0142] Under the same chromatographic conditions, the elution gradient with the highest proportion of acetonitrile is maintained, the elution time is doubled, and the chromatogram is recorded, and the results are shown below Figure 13 .

[0143] The results show that no obvious chromatographic peaks flow out after the original gradient elution is completed, indicating that the chromatographic conditions basically meet the principle of maximum information.

[0144] 5.3. Study of repeatability

[0145] Take the same batch of samples, grind finely, take about 0.5 g, accurately weigh, 6 parallel, respectively, according to the test solution preparation method in the text, to prepare sample test solution, sample analysis, to investigate the consistency of characteristic peak relative retention time and relative peak area, results Table 9.

[0146] Table 9 Repeatability test results of Plumbago zeylanica Formula Granules sample (relative retention time)

[0147]

[0148] The results show that the relative retention time of each characteristic peak is less than 1%, and the repeatability test of the method is good.

[0149] 5.4. Study of precision

[0150] Take the same batch of samples, grind, take about 0.5g, accurately weighed, prepared into test solution according to the preparation method of test solution, 6 times continuously, 1 μl each time, the consistency of the relative retention time and relative peak area of characteristic peaks was investigated. The results are shown in Table 10.

[0151] Table 10 Precision test results (relative retention time)

[0152]

[0153] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the precision was good.

[0154] 5.5 Stability investigation

[0155] Take the same batch of samples, grind, take about 0.5g, accurately weighed, prepared into test solution according to the preparation method of test solution, 1 μl was injected at 0, 2, 4, 8, 12, 18, 24h respectively, and the consistency of the relative retention time and relative peak area of characteristic peaks was investigated. The results are shown in Table 11.

[0156] Table 11 Stability test results (relative retention time)

[0157]

[0158] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the test solution was stable within 24 hours.

[0159] 5.6 Intermediate precision investigation

[0160] Take the same batch of samples, 3 test solution were prepared by two experimenters A and B according to the preparation method of test solution in the text, 1 μl was injected at different times and on different instruments respectively, and the consistency of the relative retention time and relative peak area of characteristic peaks was investigated. The results are shown in Table 12.

[0161] Table 12 Intermediate precision investigation (relative retention time)

[0162]

[0163] The results showed that the RSD of the relative retention time of each characteristic peak was less than 4%, and the intermediate precision was good.

[0164] 5.7 Durability investigation

[0165] 5.7.1 Column investigation

[0166] Take the same batch of samples, according to the preparation method of test solution in the text to prepare the test solution, respectively using Eclipse Plus C18(Agilent, 2.1mm×100mm, 1.8μm); CORTECS T3(Waters, 2.1mm×100mm, 1.6μm); Kinetex XB-C18(phenomenex, 2.1mm×100mm, 1.7μm) three different chromatographic column, to investigate its separation effect. The results are shown in Table 13, Figure 14 .

[0167] Table 13 Column investigation (relative retention time)

[0168]

[0169] The results show that the sample of Juechu formula granules has peak 2 in the CORTECS T3(Waters, 2.1mm×100mm, 1.6μm) column, and the relative retention time of peak 2 in the Kinetex XB-C18(phenomenex, 2.1mm×100mm, 1.7μm) column exceeds the specified range. Subsequent studies use Eclipse Plus C18(Agilent, 2.1mm×100mm, 1.8μm) column.

[0170] 5.7.2 Column temperature investigation

[0171] Take the same batch of samples, according to the preparation method of test solution in the text to prepare the test solution, with Eclipse Plus C18(Agilent, 2.1mm×100mm, 1.8μm) as the chromatographic column, respectively investigate the sample separation effect when the column temperature is 20℃, 25℃, 30℃. The results are shown in Table 14, Figure 15 .

[0172] Table 14 Column temperature investigation (relative retention time)

[0173]

[0174] The results show that within the range of 20℃-30℃, small changes in column temperature can meet the system suitability requirements, and subsequent studies use a column temperature of 25℃.

[0175] 5.7.3 Flow rate investigation

[0176] Take the same batch of samples, according to the preparation method of test solution in the text to prepare the test solution, investigate three flow rates of 0.25ml / min, 0.30ml / min, 0.35ml / min, record the retention time of the characteristic peaks, calculate the relative retention time according to the text requirements, the results are shown in Table 15, Figure 16 .

[0177] Table 15 Flow rate investigation (relative retention time)

[0178]

[0179] The results show that the flow rate in the range of 0.25 ml / min to 0.35 ml / min is relatively stable for the determination of the sample and meets the requirements of the method for retention time, and has good robustness.

[0180] Establishment of a method for determining the content of Jiaochu formula granules and its methodological study

[0181] 1. Instruments and reagents

[0182] Agilent high performance liquid chromatograph (1290 Infinity, Agilent Corporation); Waters high performance liquid chromatograph (Waters H-Class, Waters Corporation); one-hundredth scale balance (AP224W, Mettler-Toledo Corporation); millionth scale balance (XBR36, Mettler-Toledo Corporation); digital ultrasonic cleaner (2700TH, Shanghai Anpu Experimental Technology Co., Ltd.); electric heating constant temperature water bath (J-HH-6A, Shanghai Shengwei Electronics Technology Co., Ltd.); ultrapure water system (Comfort1, Germany Sartorius Corporation); phosphoric acid (chromatographically pure, Shanghai Maikelin Biotechnology Co., Ltd.); acetonitrile (chromatographically pure, CNW Corporation); water is ultrapure water; and other reagents are analytical pure.

[0183] Jiaochu formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0184] 2. Source of reference substance and purity check

[0185] Jiaochu ester A reference substance (batch number: PS013032) and Jiaochu ester B reference substance (batch number: PS013033) were purchased from Chengdu Pusai Biological Technology Co., Ltd. for content determination, with a content of 98%, and no treatment was required before use.

[0186] 3. Determination of detection wavelength

[0187] The spectra of Jiaochu ester A and Jiaochu ester B at 190-400 nm were collected, and the ultraviolet absorption graph was recorded, as shown in Figure 5 , Figure 6 .

[0188] The results show that Jiaochu ester A and Jiaochu ester B have strong absorption at about 260 nm, and 262 nm is finally selected as the detection wavelength.

[0189] 4. Determination of chromatographic conditions

[0190] Reference to the chromatographic profile, the following chromatographic conditions were determined: octadecylsilane-bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile-0.1% phosphoric acid solution (45:55) as the mobile phase; flow rate 0.3 ml / min; column temperature 30 °C, and detection wavelength 262 nm.

[0191] 5. Preparation of test solution

[0192] 5.1 Investigation of different extraction solvents

[0193] Take a suitable amount of the Granules of Justicia Formula, grind finely, take about 0.5 g, parallel 5 groups, 2 portions in each group, accurately weigh, place in a conical flask with a plug, accurately add water, 30% methanol, 50% methanol, 70% methanol and methanol respectively, 25 ml each, tightly plug, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, and obtain. Accurately take 1 μl of each test solution, inject into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the contents of Justicidin B and Justicidin A, the results are shown in Table 16, Figure 17 .

[0194] Table 16 Comparison of different extraction solvents

[0195]

[0196] Conclusion: When water, 30% methanol, 50% methanol, 70% methanol and methanol are used as the extraction solvent respectively, the results show that the contents of Justicidin B and Justicidin A obtained by methanol extraction are higher, therefore, methanol is selected as the extraction solvent.

[0197] 5.2 Investigation of different extraction methods

[0198] Take a suitable amount of the Granules of Justicia Formula, grind finely, take about 0.5 g, parallel 3 groups, 2 portions in each group, accurately weigh, place in a conical flask with a plug, accurately add methanol 25 ml, tightly plug, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz), heating reflux and shaking extraction respectively for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and obtain. Accurately take 1 μl of each test solution, inject into the liquid chromatograph, determine according to the above chromatographic conditions, calculate the contents of Justicidin B and Justicidin A, the results are shown in Table 17, Figure 18 .

[0199] Table 17 Comparison of different extraction methods

[0200] Conclusion: From the results, it can be seen that the extraction efficiency of ultrasonic treatment and heating reflux is close, for the convenience of experimental operation,

[0201]

[0202] Finally, the extraction method is ultrasonic treatment.

[0203] 5.3 Investigation of different extraction time

[0204] Take a proper amount of the granules of the prescription of the plant of Justicia bedfordioides, grind them finely, take about 0.5 g, divide them into 4 groups in parallel, each group has 2 portions, precisely weigh them, put them in conical flasks with stoppers, precisely add 25 ml of methanol, tightly stop them, weigh them, ultrasonic treat them (power 600 W, frequency 40 kHZ) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, cool them, weigh them again, make up the weight loss with methanol, shake them well, filter them, take the filtrate, and you get them. Precisely take 1 μl of each test solution, inject it into the liquid chromatograph, determine the content of B and A according to the above chromatographic conditions, and the results are shown in Table 18. Figure 19 .

[0205] Table 18 Comparison of different extraction time

[0206]

[0207]

[0208] Conclusion: According to the results, the content of B and A is higher when the extraction time is 45 minutes, so the extraction time is selected as 45 minutes.

[0209] 5.4 Investigation of different extraction volume

[0210] Take a proper amount, grind them finely, take about 0.5 g, divide them into 3 groups in parallel, each group has 2 portions, precisely weigh them, put them in conical flasks with stoppers, precisely add 15 ml, 25 ml and 50 ml of methanol respectively, tightly stop them, weigh them, ultrasonic treat them (power 600 W, frequency 40 kHZ) for 45 minutes, cool them, weigh them again, make up the weight loss with methanol, shake them well, filter them, take the filtrate, and you get them. Precisely take 1 μl of each test solution, inject it into the liquid chromatograph, determine the content of B and A according to the above chromatographic conditions, and the results are shown in Table 19. Figure 20 .

[0211] Table 19 Comparison of different extraction volume

[0212]

[0213] Conclusion: According to the results, the content of B and A is higher when the extraction solvent volume is 25 ml, so the solvent volume is selected as 25 ml.

[0214] After investigation, the preparation method of the test sample for the determination of the content of the particle is finally determined as follows:

[0215] Take the appropriate amount of the particle of the formula of the basil, grind it finely, take about 0.5 g, accurately weigh it, put it in a conical bottle with a stopper, accurately add 25 ml of methanol, seal it tightly, weigh it, ultrasonic treat it (power 600 W, frequency 40 kHZ) for 45 minutes, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate, which is obtained.

[0216] 6. Methodology verification

[0217] 6.1 Linearity

[0218] Accurately take 0.1 ml, 0.5 ml, 1.0 ml, 2.0 ml, and 5.0 ml of the stock solution of the control sample of the basil ester B (concentration 98.9800 μg / ml) into 10 ml volumetric flasks, add methanol to the calibration mark, shake well, and prepare control sample solutions containing 0.9898 μg, 4.9490 μg, 9.8980 μg, 19.7960 μg, and 49.4900 μg of the basil ester B per 1 ml, respectively. Accurately take the above control sample solutions and the stock solution, and sequentially inject 1 μl. Take the peak area integral value as the vertical coordinate and the injection amount (μg) as the horizontal coordinate to draw a standard curve, and obtain the regression equation of the basil ester B. The results are shown in Tables 20-22. Figure 21 Figure 22

[0219] Table 20 Regression equation

[0220]

[0221] Table 21 Relationship between the peak area integral value of the control sample of the basil ester B and the injection amount

[0222]

[0223] Table 22 Relationship between the peak area integral value of the control sample of the basil ester A and the injection amount

[0224]

[0225] Results: The injection amount of the basil ester B is in the range of 0.00099-0.09898 μg, and the injection amount of the basil ester A is in the range of 0.00100-0.09957 μg, which shows a good linear relationship with the peak area value.

[0226] 6.2 Precision test

[0227] 6.2.1 Instrument precision test

[0228] ​​Take the formula granules of Justicia bedfordiana, according to the sample preparation method of test solution in the text to prepare sample test solution, 1 μl was precisely taken and injected into liquid chromatograph, according to the above chromatographic conditions, 6 times of continuous injection were carried out, the peak area measurement values of Justicia bedfordiana ester B and Justicia bedfordiana ester A were recorded, and the RSD was calculated, and the results were shown in the following table.

[0229] Table 23 Instrument precision test

[0230]

[0231] The results showed that the instrument precision test was good.

[0232] 6.2.2 Reproducibility test

[0233] Take the formula granules of Justicia bedfordiana, about 0.5 g was finely ground, and 6 portions were precisely weighed, and sample test solution was prepared according to the sample preparation method of test solution in the text, 1 μl was injected, the peak area values of Justicia bedfordiana ester B and Justicia bedfordiana ester A were determined, and the content and RSD were calculated, and the results were shown in table 24.

[0234] Table 24 Reproducibility test results of Justicia bedfordiana formula granules sample

[0235]

[0236] Results: the RSD of the contents of Justicia bedfordiana ester B and Justicia bedfordiana ester A was less than 3%, and the reproducibility test was good.

[0237] 6.2.3 Intermediate precision test

[0238] Take the formula granules of Justicia bedfordiana, 3 test solution samples were prepared by two experimenters A and B according to the sample preparation method of test solution in the text, 1 μl was injected at different times and on different instruments, the peak area values of Justicia bedfordiana ester B and Justicia bedfordiana ester A were determined, and the content and RSD were calculated, and the results were shown in table 25 and table 26.

[0239] Table 25 Intermediate precision test (Justicia bedfordiana ester B)

[0240]

[0241] Table 26 Intermediate precision test (Justicia bedfordiana ester A)

[0242]

[0243] Results: the RSD of the contents of Justicia bedfordiana ester B and Justicia bedfordiana ester A was less than 2%, and the intermediate precision test was good.

[0244] 6.3 Accuracy test

[0245] 0.25g, a total of 9 parts, accurately weighed, added with 3 parts of 50%, 100%, and 150% of the control substances of acanthoic acid ester B and acanthoic acid ester A contained in 0.25g sample respectively, and the sample recovery test solution was prepared according to the preparation method of the test solution in the main text. According to the chromatographic conditions of the content determination in the main text, 1μl was injected respectively, and the recovery rate and RSD were calculated with the following formula. The results are shown in the table below.

[0246] Table 27 Accuracy test (Acanthoic acid B)

[0247]

[0248] Table 28 Accuracy test (Acanthoic Acid)

[0249]

[0250]

[0251] Results: The recovery rates of acanthocyanidin B were between 95.88% and 103.18%, and the recovery rates of acanthocyanidin A were between 101.72% and 106.48%, indicating a good accuracy test.

[0252] 6.4 Specificity test

[0253] The excipients added to the Acanthus chinensis formula granules are maltodextrin, silicon dioxide and magnesium stearate. This experiment investigated the effect of negative samples lacking Acanthus chinensis on the Acanthus chinensis formula granules.

[0254] Take the negative sample lacking Acanthus orchioides and prepare the negative sample solution according to the test sample preparation method.

[0255] Take 1 μl of each of the Acanthopanax formula granules, negative control solution, Acanthopanax B and Acanthopanax A reference solution and inject them according to the chromatographic conditions in the text. The results are shown below. Figure 23 .

[0256] Conclusion: The results show that the excipients and solvents have no interference in the determination of acanthoside B and acanthoside A, and the method has strong specificity.

[0257] 6.5 Integrity test

[0258] Under the same chromatographic conditions, maintain the elution gradient when the acetonitrile ratio is the highest, double the elution time, and record the chromatogram. Figure 24 .

[0259] Conclusion: The results show that doubling the analysis time does not interfere with the determination of acanthodin B and acanthodin A.

[0260] 6.6 Durability test

[0261] 6.6.1 Stability test

[0262] Take the Aconite formula granules and prepare the test solution according to the preparation method of the test solution in the main text. Inject 1 μl at 0, 2, 4, 8, 12, 18, and 24 hours respectively, measure the peak area value, and calculate its RSD. The results are shown in the table below;

[0263] Table 29 Stability test results

[0264]

[0265] The results showed that the sample solution had good stability within 24 hours.

[0266] 6.6.2 Chromatographic column inspection

[0267] The test solution was prepared by taking the granules of Acanthopanax quinata and following the preparation method of the test solution in the main text. Three different chromatographic columns, Eclipse Plus C18 (Agilent, 2.1mm×100mm, 1.8μm); CORTECS T3 (Waters, 2.1mm×100mm, 1.6μm); Kinetex XB-C18 (phenomenex, 2.1mm×100mm, 1.7μm), were used to investigate the contents of Acanthopanax quinata B and Acanthopanax quinata. The results are shown in Table 30. Figure 25 .

[0268] Table 30 Investigation of different chromatographic columns

[0269]

[0270] Results: The three different types of chromatographic columns had good separation of target components, with similar contents of acanthocyanidin B and acanthocyanidin A, and good durability.

[0271] 6.6.3 Investigation of different column temperatures

[0272] Take the formula granules of Acanthopanax, and prepare the test solution according to the preparation method of the test solution in the main text. The contents of Acanthopanax B and Acanthopanax A at three temperatures of 25℃, 30℃ and 35℃ were investigated. The results are shown in Table 31. Figure 26 .

[0273] Table 31 Investigation of different column temperatures

[0274]

[0275] Results: When the column temperature was within the range of 25℃~35℃, small changes in column temperature had no significant effect on the content determination of the sample, and the column had good durability.

[0276] 6.6.4 Investigation of different flow rates

[0277] Take valerian formula granules, according to the preparation method of test solution of the text preparation of test solution, to investigate 0.25 ml / min, 0.30 ml / min, 0.35 ml / min three flow rate under valerian ester B, valerian ester A content results are shown in table 32, Figure 27 .

[0278] Table 32 different flow rate investigation

[0279]

[0280] Results: the flow rate in the range of 0.25 ml / min ~ 0.35 ml / min, the small flow rate variation has no significant effect on the determination of the content of the sample, good durability.

[0281] 7. The determination of the content limit of formula granules according to the process research results, the content of valerian formula granules is as follows:

[0282] Table 33 different batches of valerian content results

[0283] Serial number Glabridin B content % Glabridin A content % 1 0.023 0.066 2 0.019 0.049 3 0.024 0.057 4 0.043 0.097 5 0.026 0.067 6 0.028 0.070 7 0.022 0.054 8 0.017 0.044 9 0.018 0.047 10 0.044 0.078 11 0.042 0.075 12 0.060 0.101 13 0.059 0.087 14 0.072 0.112 15 0.074 0.116 16 0.042 0.062 17 0.046 0.050 18 0.044 0.048 min ~ max 0.017～0.074 0.044～0.116 Mean value 0.039 0.071 SD 0.02 0.02 Mean value ± 30% 0.03～0.05 0.05～0.09 Mean value ± 3SD -0.02～0.09 0～0.14

[0284] According to the results of 18 batches of valerian, the average content of valerian ester B in 18 batches of valerian formula granules is 0.039%, and the SD value is 0.02%; the average content of valerian ester A is 0.071%, and the SD value is 0.02%. Considering that this index is newly established, therefore, on the basis of the average content of 18 batches of granules, the lower limit of the content of valerian ester B in valerian formula granules is 1.5 times the SD, and the calculated result is 0.012%; on the basis of the average content of 18 batches of granules, the upper limit of the content of valerian ester B in valerian formula granules is 2.5 times the SD, and the calculated result is 0.085%, the content range is revised to 0.01% ~ 0.08%. On the basis of the average content of 18 batches of granules, the lower limit of the content of valerian ester A in valerian formula granules is 2 times the SD, and the calculated result is 0.025%; on the basis of the average content of 18 batches of granules, the upper limit of the content of valerian ester A in valerian formula granules is 3 times the SD, and the calculated result is 0.140%, the content range is revised to 0.03% ~ 0.15%. The content range of valerian ester B in 1 g of valerian formula granules is 0.10 mg ~ 0.80 mg, and the content range of valerian ester A is 0.30 mg ~ 1.50 mg.

Claims

1. A method of quality control of a sample of a coleus plant, characterized by, The method comprises the following steps: (1) selecting a sample of Justicia procumbens L. and preparing a sample solution; using Justicia procumbens L. as a control medicinal material to prepare a reference solution of the control medicinal material; using Justicia procumbens L. as a control reference solution, and preparing a control reference solution of Justicia procumbens L.; (2) respectively taking the reference solution and the sample solution to perform ultra-high performance liquid chromatography analysis, and constructing a characteristic spectrum of the sample to be measured. The extraction agent of the sample solution is methanol with a volume concentration of 50-100 %, and the ultra-high performance liquid chromatography analysis is performed under the following conditions: the column is Eclipse Plus C18, the column length is 100 mm, the inner diameter is 2.1 mm, the particle size is 1.8 μm, acetonitrile is used as the mobile phase A, and a 0.05-0.2 % phosphoric acid solution is used as the mobile phase B, gradient elution is performed as follows: 0-6 min, 27→31 % A, 6-10 min, 31→38 % A, 10-15 min, 38→65 % A, 15-18 min, 65→80 % A, 18-21 min, 80 % A, 21-22 min, 80→27 % A; an ultraviolet detector is used, and the detection wavelength is 254-280 nm; the sample of Justicia procumbens L. is a medicinal material, a decoction piece or a formula granule of Justicia procumbens L. In step (1), the extraction agent for preparing the reference solution of the control medicinal material is methanol with a volume concentration of 50-100 %, and the extraction agent is used in an amount of 30-100 ml per 0.2-1.0 g of Justicia procumbens L., and then ultrasonic extraction is performed; in the preparation of the sample solution, the extraction agent is used in an amount of 15-50 ml per 0.2-1.0 g of Justicia procumbens L., and then ultrasonic extraction is performed, and the ultrasonic extraction time is 15-60 minutes. In step (1), the concentration of the control reference solution of Justicia procumbens L. is 5-50 μg / ml.

2. The method for quality control of a sample of Saintpaulia according to claim 1, characterized in that, The flow rate is 0.25-0.35 ml / min, and the column temperature is 20-30 °C.

3. The method for quality control of a sample of Saintpaulia according to claim 1, characterized in that, In the seven characteristic peaks, peak 5, peak 6 and peak 7 correspond to the retention time of the reference peak of the control reference solution of Justicia procumbens L., and the peak corresponding to the reference peak of the control reference solution of Justicia procumbens L. is the S peak; the relative retention time of each characteristic peak to the S peak is calculated; the relative retention time should be within ±10 % of the specified value, and the specified value is: peak 1: 0.38, peak 2: 0.40, peak 3: 0.61, and peak 4: 0.

72.

4. The method for quality control of a sample of Saintpaulia according to claim 1, characterized in that, The content determination of the sample of Justicia procumbens L. comprises the following steps:

5. The method for quality control of a sample of Saintpaulia according to claim 1, characterized in that, ​ ​ 6. The method for quality control of a sample of Saintpaulia according to claim 1, characterized in that, ​ (1) Select the sample of Justicia bedellonis, and prepare the test solution; take Justicia bedellonis ester A and Justicia bedellonis ester B as the reference substance, and prepare the reference substance solution; (2) Take the reference substance solution and the test solution respectively to carry out the ultra-high performance liquid chromatography analysis, and construct the content determination method of the sample to be tested, and the content index is Justicia bedellonis ester A and Justicia bedellonis ester B.

7. The method for quality control of a sample of Saintpaulia according to claim 6, characterized in that, In step (1), the extraction agent for preparing the test solution is methanol with a volume concentration of 70% to 100%, and the corresponding extraction agent usage amount is 30ml to 100ml per gram of Justicia bedellonis, and the extraction time is 15 to 60 minutes, and the concentration of Justicia bedellonis ester A and Justicia bedellonis ester B reference substance solution is 5 to 50ug / ml.

8. The method for quality control of a sample of Saintpaulia according to claim 6, characterized in that, The conditions of the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile-0.05% to 0.2% phosphoric acid solution is used as the mobile phase; the detection wavelength is 190 to 400nm; the flow rate is 0.25ml to 0.35ml per minute; the column temperature is 25 to 35 degrees Celsius; and the ultraviolet detector is used.

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