HPLC content determination and characteristic chromatogram construction method for fructus chebulae immaturus and processed product thereof

Abstract

The invention discloses an HPLC content determination and characteristic chromatogram construction method for fructus chebulae immaturus and a processed product thereof, and belongs to the technical field of traditional Chinese medicine analysis and detection.According to the method, a high performance liquid chromatography method is adopted, a C18 column with octadecylsilane chemically bonded silica as a filler is adopted, a methanol-0. 1% phosphoric acid aqueous solution is adopted as a mobile phase for gradient elution, the detection wavelength is 270 nm, the detection time is 1 h, and the detection result is accurate. The content of gallic acid, punicalagin and chebulagic acid can be measured at the same time, and a characteristic spectrum containing seven characteristic peaks is constructed. A test sample is prepared by ultrasonic extraction with 35% acetonitrile. The methodology verification shows that the method has good precision, repeatability, stability, sample recovery rate and durability, can effectively realize multi-component quantitative and characteristic spectrum qualitative analysis of the immature fruit and almond oil stir-fried product, fills the blank of the existing quality standard, and provides a simple and reliable technical means for quality control of the immature fruit and almond oil stir-fried product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis and detection, and particularly relates to a HPLC content determination and characteristic spectrum construction method of Xiqingguo and its processed products. BACKGROUND

[0002] Xiqingguo is the dried young fruit of Terminalia chebula Retz. of Combretaceae. It is bitter, sour and astringent in nature and has the functions of clearing heat and generating saliva and detoxification. It is used for treating yin deficiency and white throat. The processing of Xiqingguo is currently mostly removing impurities, or crushing, or softening and cutting, drying and other cleaning. In the processing of traditional Chinese medicine, almond oil is used to fry Xiqingguo, which can not only correct the odor, purify the medicinal materials and keep dry, but also enhance the efficacy, reduce the side effects and ensure the safety of the medicine.

[0003] At present, Xiqingguo is recorded in the Pharmacopoeia of the People's Republic of China (2025 edition), and only identification, inspection and extract are listed under it, lacking the content determination and characteristic spectrum control of the effective components, which is difficult to comprehensively evaluate the quality of medicinal materials. At the same time, there are few reports on the characteristic spectrum and content determination of Xiqingguo, and the quality research of its processed products is in a blank state.

[0004] With the development of traditional Chinese medicine, Xiqingguo and its processed products are increasingly widely used, but the lack of quality control methods leads to the difficulty in ensuring the quality stability in the production process, and it is also impossible to provide reliable basis for the effectiveness and safety of clinical medication. Therefore, it is of great significance to establish a method for simultaneously determining the content of multiple components and analyzing the characteristic spectrum of Xiqingguo and its processed products, which is important for improving the quality standard and enhancing the quality control level. SUMMARY

[0005] The present application provides a HPLC content determination and characteristic spectrum construction method of Xiqingguo and its processed products, which solves the above problems.

[0006] The present application provides a HPLC content determination and characteristic spectrum construction method of Xiqingguo and its processed products, which adopts high performance liquid chromatography to simultaneously determine the contents of gallic acid, punicalin and chebulagic acid in Xiqingguo and its processed products and construct the characteristic spectrum, including the following steps:

[0007] S1, preparation of test solution: taking Xiqingguo or its processed products, crushing, accurately weighing, adding 15-35% volume percentage of acetonitrile aqueous solution, ultrasonic treatment, taking out and cooling, constant volume, shaking, filtering, and taking the filtrate to obtain; the processed product is almond oil fried Xiqingguo;

[0008] S2, preparation of reference solution of control: respectively taking gallic acid control, punicalin control and chebulagic acid control, dissolving in 35% acetonitrile to prepare a mixed solution;

[0009] S3, Preparation of reference solution of control medicinal material: Take the control medicinal material of Terminalia chebula, accurately weigh, add 35% acetonitrile, ultrasonic treatment, cool, shake well, filter, take the filtrate to obtain;

[0010] S4, HPLC detection: inject the test solution, the reference solution of control product, the reference solution of control medicinal material into the high performance liquid chromatograph, and record the chromatogram;

[0011] The chromatographic conditions include: the chromatographic column is a C18 column with octadecylsilane bonded silica gel as the filler; the detection wavelength is 270 nm; the mobile phase A is methanol, and the mobile phase B is 0.1% phosphoric acid aqueous solution, which is eluted according to the following gradient program:

[0012] Time 0-25 min, mobile phase A 5→20%, mobile phase B 95→80%; time 25-55 min, mobile phase A 20→35%, mobile phase B 80→65%; time 55-70 min, mobile phase A 35→50%, mobile phase B 65→50%; time 70-75 min, mobile phase A 50→5%, mobile phase B 50→95%; time 75-80 min, mobile phase A 5→5%, mobile phase B 95→95%;

[0013] S5, result analysis: based on the chromatogram, the content of three components is calculated, and the test chromatogram should present seven characteristic peaks corresponding to the retention time of the seven characteristic peaks of the reference chromatogram of the control medicinal material, wherein peak 1, peak 2, peak 3, peak 5 are respectively consistent with the retention time of gallic acid, punicalin alpha, punicalin beta and chebulagic acid reference substance; peak 4 is an unknown component, peak 6 is chebulagic acid, and peak 7 is ellagic acid; peak 5 is S peak, and the relative retention time of the remaining characteristic peaks to S peak is within ±10% of the specified value, the specified value of peak 4 is 0.79, the specified value of peak 6 chebulagic acid is 1.27, and the specified value of peak 7 ellagic acid is 1.34.

[0014] Preferably, in the above-mentioned HPLC content determination and characteristic chromatogram construction method of Terminalia chebula and its processed products, the chromatographic column is a C18 column with a particle size of 5 μm and a diameter of 250 mm×4.6 mm.

[0015] Preferably, in the above-mentioned HPLC content determination and characteristic chromatogram construction method of Terminalia chebula and its processed products, the chromatographic conditions of HPLC detection further include: column temperature 33-37°C; flow rate 1.0 mL / min; injection volume 5 μL; theoretical plate number not less than 10000 calculated by chebulagic acid.

[0016] Preferably, in the HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products, the specific parameters for preparing the test solution in step S1 include: the solvent is 15%-35% acetonitrile aqueous solution; the solvent is added in an amount of 50 mL-100 mL relative to 0.1 g of the test sample; the ultrasonic treatment power is 200 W, the frequency is 40 kHz, and the time length is 20 min-40 min.

[0017] Preferably, in the HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products, the solvent is 35% acetonitrile aqueous solution, the solvent is added in an amount of 50 mL relative to 0.1 g of the test sample, and the ultrasonic treatment time length is 30 min.

[0018] Preferably, in the HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products, the concentration of the reference solution of the control sample in step S2 is: 20 μg-40 μg of gallic acid, 150 μg-170 μg of punicalin, and 240 μg-260 μg of chebulagic acid per 1 mL.

[0019] Preferably, in the HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products, the preparation parameters of the reference solution of the control medicinal material in step S3 are: about 0.1 g of Mallotus philippinensis control medicinal material is added into 50 mL of 35% acetonitrile, and ultrasonic treatment is performed for 30 min at a power of 200 W and a frequency of 40 kHz.

[0020] Preferably, in the HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products, the preparation method of the processed products is frying with almond oil.

[0021] Therefore, the HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products has the following beneficial effects:

[0022] The HPLC content determination and characteristic spectrum construction method of Mallotus philippinensis and its processed products simultaneously quantifies gallic acid, punicalin and chebulagic acid in Mallotus philippinensis and its processed products, and constructs a characteristic spectrum containing seven characteristic peaks, which makes up for the deficiency of the existing pharmacopoeia standard that only relies on identification and control of extract, especially fills the blank of quality research of processed products. The relative retention time and the allowable fluctuation range of the seven characteristic peaks are determined, five of which correspond to known components gallic acid, punicalin alpha / beta, chebulagic acid, chebulagic acid and ellagic acid, and three of which are quantified, which can comprehensively reflect the chemical composition characteristics of Mallotus philippinensis and its processed products, and provide a scientific basis for evaluating the consistency and stability of medicinal materials and processed products.

[0023] By optimizing the chromatographic conditions, such as specific gradient elution program, etc., a method is realized to simultaneously complete the content determination and characteristic spectrum construction, without the need to separately establish two sets of detection systems, which greatly saves the detection cost and time, and improves the production efficiency. The provided content detection and characteristic spectrum method precision, repeatability, stability, sample recovery rate, durability all meet the requirements of the current edition of the pharmacopoeia, the detection result is accurate and reliable, the method is without extraction, without concentration, without evaporation, simple, fast, accurate, easy to reproduce, easy to popularize and master, the method operation is simple, and has wide applicability to the test sample, is easy to popularize in production and quality control, and provides a strong support for the quality standardization of the west green fruit and its processed products.

[0024] The technical solutions of the present application are described in further detail below by means of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is the specificity spectrum of the content determination of the west green fruit medicinal material and its processed products in the present application;

[0026] Figure 2 is the standard curve of gallic acid in the present application;

[0027] Figure 3 is the standard curve of asperosaponin in the present application;

[0028] Figure 4 is the standard curve of corilagin in the present application;

[0029] Figure 5 is the specificity spectrum of the characteristic spectrum of the west green fruit medicinal material and its processed products in the present application;

[0030] Figure 6 is the characteristic comparison spectrum of the west green fruit medicinal material in the present application;

[0031] Figure 7 is the characteristic comparison spectrum of the west green fruit processed product in the present application;

[0032] Figure 8 is the characteristic spectrum superimposed spectrum of 15 batches of west green fruit in the present application;

[0033] Figure 9 is the characteristic spectrum superimposed spectrum of 15 batches of west green fruit processed products in the present application;

[0034] Figure 10 is the west green fruit HPLC control characteristic spectrum generated by the State Pharmacopoeia Committee "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System" in the present application;

[0035] Figure 11The application discloses a HPLC control characteristic spectrum of a prepared Vauquelinia seed, which is generated by using a Chinese medicine chromatographic fingerprint similarity evaluation software system of the State Pharmacopoeia Commission. DETAILED DESCRIPTION

[0036] In order to better understand the above technical solutions, the above technical solutions will be described in detail below in combination with the drawings of the specification and specific embodiments. Obviously, the described embodiments are only part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the application.

[0037] The terms used in the embodiments of the application are only for the purpose of describing specific embodiments, and are not intended to limit the application. The singular forms "a", "said" and "the" used in the embodiments of the application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise. "Multiple" generally includes at least two.

[0038] It should also be noted that the terms "include", "contain" or any other variant thereof are intended to cover non-exclusive inclusion, so that the goods or devices including a series of elements not only include those elements, but also include other elements not explicitly listed, or include elements inherent to such goods or devices. Without more limitation, the element defined by the sentence "including a…" does not exclude the presence of other identical elements in the goods or devices including the element.

[0039] The application provides a HPLC content determination and characteristic spectrum construction method of Vauquelinia seed and its processed products. The contents of gallic acid, punicalin and chebulagic acid in Vauquelinia seed and its processed products are determined and the characteristic spectrum is constructed by using high performance liquid chromatography, which comprises the following steps:

[0040] S1, preparation of test sample solution: taking Vauquelinia seed or its processed products, crushing, accurately weighing, adding 15% to 35% of acetonitrile aqueous solution, ultrasonic treatment, taking out and cooling, constant volume, shaking, filtering, and taking the filtrate to obtain; the processed products are Vauquelinia seed fried with almond oil;

[0041] S2, preparation of reference solution of control sample: taking gallic acid control sample, punicalin control sample and chebulagic acid control sample respectively, dissolving in 35% acetonitrile to prepare a mixed solution;

[0042] S3, preparation of reference solution of control medicine: taking Vauquelinia seed control medicine, accurately weighing, adding 35% acetonitrile, ultrasonic treatment, cooling, shaking, filtering, and taking the filtrate to obtain;

[0043] S4, HPLC detection: inject the test sample solution, the control sample reference solution, the control medicinal material reference solution into the high performance liquid chromatograph, and record the chromatogram;

[0044] The chromatographic conditions include: the chromatographic column is a C18 column with octadecylsilane-bonded silica gel as the filler; the detection wavelength is 270 nm; the mobile phase A is methanol, and the mobile phase B is 0.1% phosphoric acid aqueous solution, which is eluted according to the following gradient program:

[0045] Time 0-25 min, mobile phase A 5-20%, mobile phase B 95-80%; time 25-55 min, mobile phase A 20-35%, mobile phase B 80-65%; time 55-70 min, mobile phase A 35-50%, mobile phase B 65-50%; time 70-75 min, mobile phase A 50-5%, mobile phase B 50-95%; time 75-80 min, mobile phase A 5-5%, mobile phase B 95-95%;

[0046] S5, result analysis: based on the chromatogram, the contents of the three components are calculated, and the test sample chromatogram should present seven characteristic peaks corresponding to the retention time of the seven characteristic peaks of the control medicinal material reference chromatogram, wherein peak 1, peak 2, peak 3, peak 5 are respectively consistent with the retention time of the control sample peaks of gallic acid, punicalin alpha, punicalin beta and chebulagic acid; peak 4 is an unknown component, peak 6 is chebulagic acid, and peak 7 is ellagic acid; peak 5 is the S peak, and the relative retention time of the other characteristic peaks to the S peak is within ±10% of the specified value, the specified value of peak 4 is 0.79, the specified value of peak 6 chebulagic acid is 1.27, and the specified value of peak 7 ellagic acid is 1.34.

[0047] To further optimize the above technical solution, the chromatographic column is a C18 column with a particle size of 5 μm and a diameter of 250 mm x 4.6 mm.

[0048] To further optimize the above technical solution, the chromatographic conditions of HPLC detection further include: column temperature 33-37℃; flow rate 1.0 mL / min; injection volume 5 μL; theoretical plate number not less than 10000 calculated by chebulagic acid.

[0049] To further optimize the above technical solution, the specific parameters for preparing the test sample solution in step S1 include: the solvent is 15-35% acetonitrile aqueous solution; the solvent addition amount is 50-100 mL relative to 0.1 g of test sample; the ultrasonic treatment power is 200 W, the frequency is 40 kHz, and the time length is 20-40 min.

[0050] To further optimize the above technical solution, the solvent is 35% acetonitrile aqueous solution, the solvent addition amount relative to 0.1 g of test sample is 50 mL, and the ultrasonic treatment time length is 30 min.

[0051] To further optimize the above technical solution, the concentration of the reference solution of the control in step S2 is: 20-40 μg of gallic acid, 150-170 μg of punicalin, and 240-260 μg of chebulagic acid per 1 mL.

[0052] To further optimize the above technical solution, the preparation parameters of the reference solution of the control medicinal material in step S3 are: about 0.1 g of the control medicinal material of Terminalia chebula is taken, 50 mL of 35% acetonitrile is added, ultrasonic treatment is performed for 30 min, the power is 200 W, and the frequency is 40 kHz.

[0053] To further optimize the above technical solution, the preparation method of the processed product is frying with almond oil.

[0054] To further optimize the above technical solution, the preparation method of the processed product is frying with almond oil.

[0055] Example 1

[0056] To determine the optimal preparation process of the test sample solution of Terminalia chebula and its processed products, three key factors, i.e., extraction solvent, extraction liquid ratio, and extraction time, are investigated in this embodiment.

[0057] 0.1% phosphoric acid solution preparation: 2.0 mL of phosphoric acid is taken, placed in a 2000 mL volumetric flask, diluted with purified water to the calibration mark, shaken, filtered through a 0.45 μm filter membrane, and ultrasonically treated for 20 min to obtain a 0.1% phosphoric acid solution.

[0058] Preparation of the control solution: an appropriate amount of gallic acid, punicalin, and chebulagic acid controls is accurately weighed, dissolved and diluted with 35% acetonitrile to obtain a mixed standard solution with gallic acid, punicalin, and chebulagic acid at 29.413, 130.8915, and 138.603 μg / mL, respectively.

[0059] Preparation of the control stock solution: 10.52 mg of punicalin, 16.63 mg of chebulagic acid, and 10 mL of the gallic acid control stock solution (221.796 μg / mL) are taken and placed in a 20 mL volumetric flask, dissolved and diluted with 35% acetonitrile to obtain a three-component mixed standard stock solution with gallic acid, punicalin, and chebulagic acid at 110.898 μg / mL, 516.6898 μg / mL, and 817.2814 μg / mL, respectively.

[0060] Preparation of series of control solution: accurately weigh the control samples of gallic acid, punicalin and chebulagic acid, dissolve in 35% acetonitrile to make the three-component mixed standard stock solution of gallic acid 110.898 μg / mL, punicalin 516.6898 μg / mL and chebulagic acid 817.2814 μg / mL; take 1.0, 1.8, 2.6, 3.4, 4.2, 5.0 mL of the three-component mixed standard stock solution into a 10 mL volumetric flask, and add 35% acetonitrile to constant volume to obtain the control sample mixed standard solution of series of concentrations.

[0061] Extraction solvent investigation: take about 0.1 g of the same batch of Terminalia chebula and processed products powder, accurately weigh and determine, two samples in each group, respectively, add 15% acetonitrile, 25% acetonitrile, 35% acetonitrile, ultrasonic treatment for 30 minutes (40 kHz; 200 W), cool and constant volume to the mark, shake well, take the supernatant through a 0.22 μm filter membrane to obtain the test sample solution.

[0062] Determination was carried out according to the following chromatographic conditions: C18 column with octadecylsilane bonded silica gel as the filler; detection wavelength 270 nm; mobile phase A: methanol, mobile phase B: 0.1% phosphoric acid aqueous solution, eluted according to the following gradient program: time 0-25 min, mobile phase A 5→20%, mobile phase B 95→80%; time 25-55 min, mobile phase A 20→35%, mobile phase B 80→65%; time 55-70 min, mobile phase A 35→50%, mobile phase B 65→50%; time 70-75 min, mobile phase A 50→5%, mobile phase B 50→95%; time 75-80 min, mobile phase A 5→5%, mobile phase B 95→95%; record the contents of gallic acid, punicalin and chebulagic acid.

[0063] The results show that the theoretical plate number, separation degree and tailing factor of 15%, 25% and 35% acetonitrile solutions meet the requirements, but the extraction rate of each component is the highest in 35% acetonitrile. Among them, the extraction content of gallic acid in Terminalia chebula is 1.8976% in 35% acetonitrile, punicalin is 8.0754%, and chebulagic acid is 13.4479%; in processed products, gallic acid is 1.7951%, punicalin is 6.7228%, and chebulagic acid is 14.0956%, as shown in Table 1. Therefore, 35% acetonitrile is determined as the best extraction solvent.

[0064] Table 1 Investigation results of different extraction solvents of Terminalia chebula and processed products

[0065]

[0066] Extraction ratio investigation: 0.1 g, 0.15 g, 0.20 g of the same batch of Terminalia chebula and processed product powder were precisely weighed and placed in a 100 mL volumetric flask. Appropriate amount of 35% acetonitrile was added. After ultrasonic treatment, the content was determined. The results showed that the RSD of the content of each index component was less than 2.0% under different extraction ratios, and the difference was not large. From the perspective of saving reagents, the extraction ratio of 1:500 (i.e. 0.2 g of sample added to 100 mL of solvent) was selected. At this time, the content of gallic acid in Terminalia chebula was 1.8560%, anwa-lycopene was 8.6059%, and chebulagic acid was 13.9525%; the content of gallic acid in processed products was 1.7907%, anwa-lycopene was 6.9162%, and chebulagic acid was 14.0664%, as shown in Table 2.

[0067] Table 2 Investigation results of different extraction ratios of Terminalia chebula and processed products

[0068]

[0069]

[0070] Extraction time investigation: about 0.1 g of the same batch of sample powder was precisely weighed and placed in a 100 mL volumetric flask. Appropriate amount of 35% acetonitrile was added. Ultrasonic treatment was performed for 20 min, 30 min, and 40 min, respectively. The rest of the operation was the same as above. The determination results showed that the extraction time of 20-40 min had little effect on the content of index components, and the RSD was less than 2.0%. Considering the extraction efficiency and completeness, 30 min was selected as the extraction time. At this time, the content of gallic acid in Terminalia chebula was 1.8570%, anwa-lycopene was 8.7246%, and chebulagic acid was 13.8439%; the content of gallic acid in processed products was 1.7985%, anwa-lycopene was 6.6819%, and chebulagic acid was 13.8563%, as shown in Table 3.

[0071] Table 3 Investigation results of different extraction times of Terminalia chebula and processed products

[0072]

[0073] In summary, the optimal preparation method of sample solution is as follows: about 0.1 g of Terminalia chebula or its processed product powder (passed through a No. 3 sieve) was precisely weighed and placed in a 50 mL volumetric flask. 50 mL of 35% acetonitrile was added. Ultrasonic treatment was performed at a power of 200 W and a frequency of 40 kHz for 30 minutes. After cooling, the solution was diluted to the mark with 35% acetonitrile, shaken well, filtered, and the filtrate was obtained.

[0074] Example 2

[0075] The present example was determined under the following chromatographic conditions: a C18 column with octadecylsilane-bonded silica gel as the filler; a detection wavelength of 270 nm; a mobile phase A of methanol and a mobile phase B of 0.1% phosphoric acid solution, eluted according to the following gradient program: time 0-25 min, mobile phase A 5→20%, mobile phase B 95→80%; time 25-55 min, mobile phase A 20→35%, mobile phase B 80→65%; time 55-70 min, mobile phase A 35→50%, mobile phase B 65→50%; time 70-75 min, mobile phase A 50→5%, mobile phase B 50→95%; time 75-80 min, mobile phase A 5→5%, mobile phase B 95→95%; the content determination method of Terminalia franchetiana and its processed products was comprehensively verified, including specificity, precision, repeatability, sample addition recovery, linearity, stability and durability.

[0076] Solution preparation: 0.1% phosphoric acid solution preparation: take 2.0 mL of phosphoric acid, place it in a 2000 mL volumetric flask, dilute to the mark with purified water, shake well, pass through a 0.45 μm filter membrane, and ultrasonic for 20 minutes to obtain.

[0077] Reference mixed standard solution: accurately weigh 2 appropriate amounts of gallic acid, punicalagin and chebulagic acid reference substances, add 35% acetonitrile solution to obtain 30.5793, 153.7708 and 174.5336 μg / mL reference mixed standard solutions.

[0078] Terminalia franchetiana reference material solution: take about 0.1 g of Terminalia franchetiana reference material, place it in a 50 mL volumetric flask, add an appropriate amount of 35% acetonitrile, ultrasonic for 30 min, cool, add 35% acetonitrile to the mark, take the supernatant and pass it through a 0.22 μm filter membrane to obtain.

[0079] Preparation of repeatability of test sample solution: take 6 portions of the same batch of Terminalia franchetiana medicinal material and processed product powder (pass through a No. 3 sieve) about 0.1 g, place it in a 50 mL volumetric flask, add an appropriate amount of 35% acetonitrile, ultrasonic for 30 min, cool, add 35% acetonitrile to the mark, take the supernatant and pass it through a 0.22 μm filter membrane to obtain.

[0080] Preparation of sample recovery rate Test sample solution: 5.9 mL of gallic acid control sample stock solution (221.796 μg / mL), 5.5 mL of punicalin stock solution (1006.8011 μg / mL), and 8.4 mL of acacia acid control sample stock solution (1189.309 μg / mL) were placed in a 25 mL volumetric flask, and 35% acetonitrile was added to the mark line to obtain a control sample mixed standard solution (for spiking); about 0.1 g of Terminalia chebula Reic. powder (passed through a No. 3 sieve) was weighed into a 50 mL volumetric flask, and 35% acetonitrile was added as appropriate, and after ultrasonic treatment for 30 min, the solution was allowed to cool, and 35% acetonitrile was added to the mark line to make up the volume; 5 mL was transferred into a 10 mL volumetric flask, 3 mL of the control sample mixed standard solution (for spiking) was added, and 35% acetonitrile was added to the mark line to make up the volume, and the mixture was mixed and filtered through a 0.22 μm filter to obtain the solution.

[0081] Preparation of stability test sample solution: about 0.1 g of Terminalia chebula Reic. and processed product powder (passed through a No. 3 sieve) was weighed into a 50 mL volumetric flask, and 35% acetonitrile was added as appropriate, and after ultrasonic treatment for 30 min, the solution was allowed to cool, and 35% acetonitrile was added to the mark line to make up the volume, and the supernatant was filtered through a 0.22 μm filter to obtain the solution.

[0082] Specificity test: the elution solvent (no sample was added), the extraction solvent (35% acetonitrile), the Terminalia chebula Reic. control drug material solution, the Terminalia chebula Reic. sample solution, the Terminalia chebula Reic. processed product solution, and the control sample mixed standard were sequentially injected into the single needle, and the chromatogram was recorded, as shown in Figure 1. Figure 1 The results showed that the Terminalia chebula Reic. drug material and processed product and the control drug material had peaks at the same retention time, and the elution solvent and the extraction solvent had no corresponding chromatographic peaks. Through the identification of the control sample mixed standard, the retention times of gallic acid (t = 10.147), punicalin α (t = 16.154), punicalin β (t = 22.291), and acacia acid (t = 49.922) were consistent with those of the control sample, as shown in Table 4, indicating that the method had good specificity.

[0083] Table 4 Specificity determination results of Terminalia chebula Reic. drug material and processed product content determination

[0084]

[0085] Precision test: the control sample mixed standard was continuously injected into six needles, and the peak area RSD was calculated. The peak area RSD of gallic acid was 0.17%, that of punicalin was 1.69%, and that of acacia acid was 0.25%, as shown in Table 5, all less than 2.0%, and the instrument precision met the requirements.

[0086] Table 5 Investigation results of Terminalia chebula Reic. drug material and processed product precision

[0087]

[0088] Repeatability test: 6 portions of the same batch of Terminalia chebula Rehder et Wils. and processed products powder were weighed, treated and determined according to the preparation method of test solution. The results showed that the content of gallic acid in Terminalia chebula Rehder et Wils. was 1.6456%-1.6735%, RSD 0.78%; anoceridine was 7.3048%-7.6465%, RSD 1.64%; chebulagic acid was 12.4523%-12.8102%, RSD 0.99%. The content of gallic acid in processed products was 1.5955%-1.6343%, RSD 0.83%; anoceridine was 6.3899%-6.4992%, RSD 0.61%; chebulagic acid was 13.3933%-13.5708%, RSD 0.50%, as shown in Table 6, the repeatability was good.

[0089] Table 6 Investigation results of repeatability of Terminalia chebula Rehder et Wils. and processed products

[0090]

[0091] Recovery rate test: about 0.1 g of Terminalia chebula Rehder et Wils. powder was accurately weighed, a certain amount of mixed standard solution (for standard addition) was added, treated according to the preparation method of test solution, and the recovery rate was determined. The results showed that the average recovery rate of gallic acid in Terminalia chebula Rehder et Wils. was 99.35%, RSD 1.02%; the average recovery rate of anoceridine was 96.40%, RSD 3.04%; the average recovery rate of chebulagic acid was 100.39%, RSD 1.18%. The average recovery rate of gallic acid in processed products was 93.72%, RSD 0.32%; the average recovery rate of anoceridine was 101.41%, RSD 1.42%; the average recovery rate of chebulagic acid was 97.81%, RSD 0.55%, as shown in Tables 7-9, which met the requirements of recovery rate limit.

[0092] Table 7 Recovery rate chromatographic results of gallic acid in Terminalia chebula Rehder et Wils. and processed products

[0093]

[0094] Table 8 Recovery rate chromatographic results of anoceridine in Terminalia chebula Rehder et Wils. and processed products

[0095]

[0096]

[0097] Table 9 Recovery rate chromatographic results of chebulagic acid in Terminalia chebula Rehder et Wils. and processed products

[0098]

[0099] Linear relationship investigation: 6 series of concentrations of control sample mixed standard were taken into sample, with content as abscissa and peak area as ordinate to draw standard curve. Gallic acid was in the range of 11.0898-55.4490 μg / mL, regression equation A=16.496C-28.393, R 2 =1; punicalagin was in the range of 51.6670-258.3449 μg / mL, regression equation A=11.933C-251.17, R 2 =0.9998; acacia acid was in the range of 81.7281-408.6407 μg / mL, regression equation A=8.8216C-114.08, R 2 =1, as shown in Table 10 and Table 10, the linear relationship is good. Figures 2-4

[0100] Table 10 Linear results of gallic acid, punicalagin and acacia acid

[0101]

[0102]

[0103] Stability test: the control sample solution and the test sample solution were taken into sample at 0, 3, 6, 9, 12, 16, 20, 24 h, and the peak area RSD was calculated. The peak area RSD of gallic acid, punicalagin and acacia acid in the control sample mixed standard was 0.13%, 0.38% and 0.39% respectively; the peak area RSD of the three in the crude drug of Terminalia chebula was 0.13%, 0.35% and 0.19% respectively; and the peak area RSD of the three in the processed product was 0.18%, 0.23% and 0.16% respectively, as shown in Table 11, all less than 2.0%, indicating that the solution was stable within 24 h.

[0104] Table 11 Investigation results of 24 h stability of crude drug of Terminalia chebula, processed product and control

[0105]

[0106]

[0107] ​Durability test: the influence of different column temperature (33℃, 35℃, 37℃) and different batch chromatographic column (Agilent 5TC-C18, SN: 736052, 570628, 715857, 738946) on the determination results was investigated. In the column temperature test, the RSDs of gallic acid, punicalin, and chebulagic acid in the medicinal material of Terminalia chebula were 1.32%, 1.81%, and 0.40%, respectively; and those in the processed product were 1.26%, 1.63%, and 0.60%, respectively, as shown in Table 12. In the different chromatographic column test, the RSDs of gallic acid, punicalin, and chebulagic acid in the medicinal material were 2.35%, 3.74%, and 1.67%, respectively; and those in the processed product were 1.13%, 2.60%, and 1.24%, respectively, as shown in Table 13, all of which met the requirements, and the method was good in durability.

[0108] Table 12 Investigation results of column temperature for the durability of Terminalia chebula medicinal material and processed product

[0109]

[0110] Table 13 Information table of chromatographic column for the durability of Terminalia chebula medicinal material and processed product

[0111] Chromatographic column Model Lot number Chromatographic column 1 Agilent 5TC-C18(2); 5 μm; 4.6 x 250 mm SN: 736052 Chromatographic column 2 Agilent 5TC-C18(2); 5 μm; 4.6 x 250 mm SN: 570628 Chromatographic column 3 Agilent 5TC-C18(2); 5 μm; 4.6 x 250 mm SN: 715857 Chromatographic column 4 Agilent 5TC-C18(2); 5 μm; 4.6 x 250 mm SN: 738946

[0112] Example 3

[0113] In this example, the characteristic chromatograms of Terminalia chebula and its processed products were constructed and the method validation was performed.

[0114] Solution preparation: 0.1% phosphoric acid solution was prepared by taking 2 mL of phosphoric acid, diluting with purified water to 2000 mL, shaking well, passing through a 0.45 μm filter membrane, and ultrasonicating for 20 minutes.

[0115] Controlled substance mixed standard solution: appropriate amounts of gallic acid, punicalin, chebulagic acid, and chebulagic acid controlled substances were weighed and dissolved in 35% acetonitrile to obtain a controlled substance stock solution mixed standard; appropriate amounts of ellagic acid controlled substance were weighed and dissolved in methanol to obtain a controlled substance stock solution; before use, appropriate amounts of the 4-component mixed standard stock solution and the ellagic acid stock solution were taken, and 35% acetonitrile was added to the mark to obtain a 5-component controlled substance mixed standard solution with concentrations of gallic acid, punicalin, chebulagic acid, ellagic acid, and chebulagic acid of 30, 150, 250, 60, and 100 μg / mL, respectively.

[0116] Terminalia chebula controlled medicinal material solution: about 0.1 g of Terminalia chebula controlled medicinal material was weighed and placed in a 50 mL volumetric flask, appropriate amount of 35% acetonitrile was added, and after ultrasonicating for 30 min, it was cooled, 35% acetonitrile was added to the mark, and the supernatant was filtered through a 0.22 μm filter membrane to obtain it.

[0117] Preparation of specific and precise test solution: Weigh 0.1g of Terminalia chebula powder and 0.1g of processed Terminalia chebula powder and place them in a 50mL volumetric flask. Add an appropriate amount of 35% acetonitrile, sonicate for 30min, cool, and add 35% acetonitrile to make up to the mark. Take the supernatant and filter it through a 0.22μm filter membrane to obtain the test solution.

[0118] Preparation of repeatable test solution: Weigh about 0.1g of 6 portions of the same batch of Terminalia chebula medicinal material and processed product powder (passed through No. 3 sieve), place them in a 50mL volumetric flask, add an appropriate amount of 35% acetonitrile, sonicate for 30min, cool, add 35% acetonitrile to make up to the mark, and filter the supernatant through a 0.22μm filter membrane to obtain the solution.

[0119] Preparation of stability test solution: Weigh about 0.1g of the powder of Terminalia chebula medicinal material and processed product (passed through No. 3 sieve), place it in a 50mL volumetric flask, add an appropriate amount of 35% acetonitrile, sonicate for 30min, cool, add 35% acetonitrile to make up to the mark, and filter the supernatant through a 0.22μm filter membrane to obtain the solution.

[0120] Preparation of the durability test solution: Weigh about 0.1 g of the powdered and processed *Terminalia chebula* (passed through a No. 3 sieve), place it in a 50 mL volumetric flask, add an appropriate amount of 35% acetonitrile, sonicate for 30 min, cool, add 35% acetonitrile to the mark, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution.

[0121] The determination was performed under the chromatographic conditions described below. The chromatographic column was a C18 column packed with octadecylsilane-bonded silica gel; the detection wavelength was 270 nm; mobile phase A was methanol, and mobile phase B was 0.1% aqueous phosphoric acid solution. Elution was performed according to the following gradient program: time 0–25 min, mobile phase A 5→20%, mobile phase B 95→80%; time 25–55 min, mobile phase A 20→35%, mobile phase B 80→65%; time 55–70 min, mobile phase A 35→50%, mobile phase B 65→50%; time 70–75 min, mobile phase A 50→5%, mobile phase B 50→95%; time 75–80 min, mobile phase A 5→5%, mobile phase B 95→95%; the chromatogram was recorded.

[0122] Characteristic chromatogram construction: Fifteen batches of Terminalia chebula and fifteen batches of processed Terminalia chebula were prepared and analyzed to obtain characteristic chromatograms for each batch. The chromatograms of these fifteen batches of Terminalia chebula and processed products were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for data processing. Similarity was calculated through peak matching, and overlay chromatograms were generated, as shown in the attached figure. Figures 8-9 A control chromatogram was generated using the mean method, such as... Figures 10-11The results show that the test sample chromatogram presents 7 characteristic peaks, which correspond to the retention time of the 7 characteristic peaks of the reference chromatogram of the control medicinal material. Among them, peak 1 is gallic acid, peak 2 is punicalin alpha, peak 3 is punicalin beta, peak 5 is chebulagic acid (S peak), peak 6 is chebulagic acid, and peak 7 is ellagic acid. Taking peak 5 as the S peak, the relative retention times of the remaining characteristic peaks are calculated, and the specified values of peak 4 (0.79), peak 6 (1.27), and peak 7 (1.34) are within the range of ±10% of the actual measured values.

[0123] Methodology verification: specificity test: sample was injected in the order of elution solvent, extraction solvent, control mixture, control medicinal material of Psidium fruit, Psidium fruit, and processed Psidium fruit. The results show that the characteristic peaks are consistent with the peaks of the control and the control medicinal material, and the blank has no interference, as shown in Table 13. Figure 5 and Table 13. The control medicinal material, the control, and the blank of Psidium fruit and processed Psidium fruit were compared, and finally 7 chromatographic peaks were determined as common peaks of the characteristic chromatogram, and 6 chromatographic peaks were identified by comparison, among which chebulagic acid was used as the reference peak S peak.

[0124] Table 13 Specificity determination results of Psidium fruit and processed Psidium fruit

[0125]

[0126] Precision test: Psidium fruit solution and processed Psidium fruit solution were injected continuously for 6 times, and the RSD% of relative retention time and relative peak area was calculated, as shown in Tables 14-19. The RSD% of retention time / relative retention time and peak area / relative peak area of the 7 chromatographic peaks of Psidium fruit and processed Psidium fruit were all less than 2.0%, indicating that the precision of the characteristic chromatogram of Psidium fruit and processed Psidium fruit was good.

[0127] Table 14 Precision determination results of characteristic chromatogram peak 1 (gallic acid)

[0128]

[0129] Table 15 Precision determination results of characteristic chromatogram peaks 2 and 3 (punicalin alpha and beta)

[0130]

[0131] Table 16 Precision determination results of characteristic chromatogram peak 4 (unknown peak)

[0132]

[0133]

[0134] Table 17 Precision determination results of characteristic chromatogram peak 5 (chebulagic acid) (S)

[0135]

[0136] Table 18 Precision test results of characteristic peak 6 (chebulagic acid)

[0137]

[0138] Table 19 Precision test results of characteristic peak 7 (ellagic acid)

[0139]

[0140]

[0141] Repeatability test: The test sample was injected once, and the RSD% of the retention time was calculated. As shown in Table 20, the retention time and the RSD% of the relative retention time of the 7 characteristic peaks in the Xiqingguo medicinal material and processed product were less than 2.0%, and the relative retention time of the unknown peak 4 with respect to the reference peak 5 was within 0.79±10% fluctuation. This indicates that the repeatability of the chromatographic condition is good.

[0142] Table 20 Repeatability test results of the characteristic chromatogram of Xiqingguo medicinal material and processed product

[0143]

[0144] Stability test: The Xiqingguo medicinal material solution and the Xiqingguo processed product solution were injected at 0, 3, 6, 9, 12, 16, 20, and 24 h, and the RSD% of the peak area was calculated. As shown in Table 21, the retention time and the RSD% of the relative retention time of the 7 characteristic peaks were less than 2.0% within 24 h, and the relative retention time of the unknown peak 4 with respect to the reference peak 5 was within 0.79±10% fluctuation within 24 h. This indicates that the 24 h stability of the Xiqingguo medicinal material solution and the Xiqingguo processed product solution under this chromatographic condition is good.

[0145] Table 21 24 h stability of the characteristic chromatogram of Xiqingguo medicinal material and processed product

[0146]

[0147] Column temperature durability test: The column temperature was set to 33°C, 35°C, and 37°C, and the reference was injected continuously for three times at each temperature, and the test sample was injected once. As shown in Table 22, the content RSD% of gallic acid, punicalin, and chebulagic acid in the Xiqingguo medicinal material and processed product was less than 2.0% at 33°C, 35°C, and 37°C, indicating that the durability of this chromatographic condition is good at 33-37°C.

[0148] Table 22 Durability column temperature investigation results of the characteristic chromatogram of Xiqingguo medicinal material and processed product

[0149]

[0150] Chromatography durability test: column temperature was set at 33℃, 35℃, 37℃, and the control sample was injected continuously for three times at each temperature, and the test sample was injected for single needle for double samples. The column information is shown in Table 13, and the results are shown in Table 23 and Figure 7 The relative retention time RSD% of each characteristic peak in the chromatographic column of different batches of Agilent 5TC-C18(2) was less than 2.0%, indicating that the chromatographic condition was good in the durability of the Agilent 5TC-C18(2) chromatographic column of different batches.

[0151] Table 23 Chromatographic column investigation results of the characteristic chromatogram of Uncariae Ramulus Cum Uncis medicinal materials and processed products

[0152]

[0153] Therefore, the present application provides the above-mentioned Uncariae Ramulus Cum Uncis and its processed products HPLC content determination and characteristic chromatogram construction method, which has the following beneficial effects:

[0154] For the first time, the simultaneous quantification of gallic acid, ampelopsin and chebulagic acid in Uncariae Ramulus Cum Uncis and its processed products, and the construction of a characteristic chromatogram containing 7 characteristic peaks, make up for the shortcomings of the existing pharmacopoeia standard which only relies on identification and extract control quality, especially fill the blank of processed product quality research. The relative retention time and allowable fluctuation range of the 7 characteristic peaks are determined, among which 5 characteristic peaks correspond to known components gallic acid, ampelopsin α / β, chebulagic acid, chebulagic acid and ellagic acid, and three of them are quantified, which can comprehensively reflect the chemical composition characteristics of Uncariae Ramulus Cum Uncis and its processed products, and provide a scientific basis for evaluating the consistency and stability of medicinal materials and processed products.

[0155] By optimizing the chromatographic conditions, such as specific gradient elution program, a method is realized to simultaneously complete content determination and characteristic chromatogram construction, without the need to establish two separate detection systems, which greatly saves the detection cost and time, and improves the production efficiency. The precision, repeatability, stability, sample recovery rate and durability of the provided content detection and characteristic chromatogram method meet the requirements of the current edition of the pharmacopoeia, and the detection results are accurate and reliable. The method is simple, fast, accurate, easy to reproduce, easy to popularize and master, and has wide applicability to the test sample, and is easy to popularize in production and quality control, which provides a strong support for the quality standardization of Uncariae Ramulus Cum Uncis and its processed products.

[0156] It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products, characterized in that, The contents of gallic acid, punicin, and chebulic acid in Terminalia chebula and its processed products were simultaneously determined by high performance liquid chromatography (HPLC), and characteristic chromatograms were constructed. The steps included: S1. Preparation of the test solution: Take Terminalia chebula or its processed product, crush it, accurately weigh it, add acetonitrile aqueous solution with a volume percentage of 15% to 35%, sonicate it, take it out and cool it, make up the volume, shake it well, filter it, and take the filtrate to obtain the test solution; the processed product is Terminalia chebula roasted with almond oil. S2. Preparation of reference solutions: Gallic acid reference standard, punicin reference standard, and chebulic acid reference standard were dissolved in 35% acetonitrile to prepare a mixed solution. S3. Preparation of reference solution for reference medicinal material: Take the reference medicinal material of Terminalia chebula, accurately weigh it, add 35% acetonitrile, sonicate, cool, shake well, filter, and take the filtrate to obtain the solution. S4. HPLC detection: Inject the test solution, reference solution, and reference medicinal material solution into the high-performance liquid chromatograph and record the chromatogram; Chromatographic conditions included: a C18 column packed with octadecylsilane-bonded silica gel; a detection wavelength of 270 nm; mobile phase A being methanol and mobile phase B being a 0.1% aqueous phosphoric acid solution, eluted using the following gradient program: Time 0–25 min, mobile phase A 5→20%, mobile phase B 95→80%; Time 25–55 min, mobile phase A 20→35%, mobile phase B 80→65%; Time 55–70 min, mobile phase A 35→50%, mobile phase B 65→50%; Time 70–75 min, mobile phase A 50→5%, mobile phase B 50→95%; Time 75–80 min, mobile phase A 5→5%, mobile phase B 95→95%; S5. Result Analysis: The content of the three components was calculated based on the chromatogram. The chromatogram of the test sample should show 7 characteristic peaks, which correspond to the retention times of the 7 characteristic peaks of the reference medicinal material. Among them, peaks 1, 2, 3, and 5 are consistent with the retention times of the reference peaks of gallic acid, punicalin α, punicalin β, and chebulic acid, respectively. Peak 4 is an unknown component, peak 6 is chebulic acid, and peak 7 is ellagic acid. Peak 5 is designated as peak S. The relative retention times of the other characteristic peaks and peak S are within ±10% of the specified values. The specified values ​​for peak 4 are 0.79, peak 6 for chebulic acid are 1.27, and peak 7 for ellagic acid are 1.

34.

2. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 1, characterized in that, The chromatographic column is a C18 column with a diameter of 250 mm × 4.6 mm and a particle size of 5 μm.

3. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 1, characterized in that, The chromatographic conditions for HPLC detection also include: column temperature 33℃~37℃; flow rate 1.0mL / min; injection volume 5μL; and theoretical plate number not less than 10000 calculated based on chebulic acid.

4. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 1, characterized in that, The specific parameters for preparing the test solution in step S1 include: the solvent is a 15%–35% acetonitrile aqueous solution; the amount of solvent added relative to 0.1g of test sample is 50mL–100mL; the ultrasonic treatment power is 200W, the frequency is 40kHz, and the duration is 20min–40min.

5. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 4, characterized in that, The solvent is a 35% acetonitrile aqueous solution, and the amount of solvent added relative to 0.1g of the test sample is 50mL. The ultrasonic treatment time is 30min.

6. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 1, characterized in that, The concentration of the reference solution in step S2 is as follows: each 1 mL contains 20 μg to 40 μg of gallic acid, 150 μg to 170 μg of pungent glycoside, and 240 μg to 260 μg of chebulic acid.

7. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 1, characterized in that, The preparation parameters for the reference solution of the control medicinal material in step S3 are as follows: take about 0.1g of Terminalia chebula reference medicinal material, add 50mL of 35% acetonitrile, and sonicate for 30min at a power of 200W and a frequency of 40kHz.

8. The method for HPLC content determination and characteristic chromatogram construction of Terminalia chebula and its processed products according to claim 1, characterized in that, The prepared product is made by roasting almond kernels in oil.

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