A method for determining five components in rhamnaceae medicinal materials based on quantitative analysis of multi-components by single marker

Abstract

The present application relates to the technical field of component detection, in particular to a method for determining five components in Rhamnaceae medicinal materials based on QAMS (quantitative analysis of multi-components by single marker). The present application creates a method for simultaneously quantitatively detecting five components in Rhamnaceae medicinal materials based on QAMS, and the contents of the five component index components are simultaneously determined by QAMS. Only one control product of chebui acid is used as an internal reference, and the specific contents of chebulagic acid, 1,2,3,6-O-tetragalloyl glucose, 1,2,3,4,6-O-pentagalloyl glucose and ellagic acid can be calculated through relative correction factors. The present application has the advantages of simple operation, high sensitivity, accurate determination and strong applicability.

Description

Technical Field

[0001] This invention relates to the field of component detection technology, and in particular to a method for determining five components in Combretaceae medicinal materials based on a one-test-multiple-evaluation method. Background Technology

[0002] *Terminalia*, belonging to the family Combretaceae, comprises approximately 200 species worldwide, widely distributed in the tropical regions of both hemispheres. Eight species are found in China: *Terminalia hainanensis*, *Terminalia chebula*, *Terminalia yunnanensis*, *Terminalia chebula*, *Terminalia peltatum*, *Terminalia scutellatus*, and *Terminalia chebula*. Also known as *Terminalia chebula*, *Terminalia chebula* fruit, and *Terminalia chebula* (in the Dai language), it is listed in the 2020 edition of the *Pharmacopoeia of the People's Republic of China* as the dried, mature fruit of *Terminalia chebula* Retz. or *Terminalia chebula* Retz. var. *tomentella* Kurt. *Terminalia chebula* is neutral in nature, bitter, sour, and astringent in taste, and enters the lung and large intestine meridians. Its functions include astringing the intestines to stop diarrhea, astringing the lungs to stop cough, reducing heat and relieving sore throat. It is mainly used to treat chronic diarrhea and dysentery, hematochezia and rectal prolapse, lung deficiency with wheezing and cough, persistent cough, sore throat, and hoarseness. As a commonly used variety in traditional Chinese medicine, Terminalia chebula is considered the "King of Tibetan Medicine." Modern research shows that its chemical components mainly include phenolic acids, tannins, triterpenoids, and flavonoids, with phenolic acids and tannins being its main active ingredients. It possesses biological activities such as antibacterial, antioxidant, antitumor, anti-inflammatory, detoxifying, analgesic, cardiotonic, hepatoprotective, hypoglycemic, inhibiting gastric emptying and small intestinal motility, promoting angiogenesis, and protecting the brain and liver. It can be used to treat various diseases and plays an important therapeutic role in several compound preparations such as Ten-Flavor Terminalia Chebula Tablets, Five-Flavor Terminalia Chebula Capsules, and Ten-Flavor Terminalia Chebula Decoction Powder. Terminalia chebula fruit, also known as Tibetan green fruit, is the dried young fruit of Terminalia chebula (Terminalia chebula Retz.), a plant in the Combretaceae family. It has the effects of clearing heat and promoting body fluid production, and detoxifying. Clinically, it is used for diphtheria due to yin deficiency and has anti-inflammatory, antibacterial, antioxidant, and hypoglycemic effects. Existing preparations mainly include Terminalia chebula granules and Tibetan green fruit throat lozenges. Terminalia bellirica (Gaertn.) Roxb., a different species within the same genus and family as Terminalia chebula, is the dried, ripe fruit of the plant. Also known as Junbaonai, Mohe, and Cimuxi, it has a sweet and astringent taste and neutral properties. It possesses the effects of clearing heat and detoxifying, astringing and nourishing blood, and harmonizing other medicines. It can be used for various heat-related syndromes, diarrhea, and liver and gallbladder diseases. Terminalia bellirica is rich in chemical components, including phenolic acids, tannins, flavonoids, and terpenes. Modern pharmacological studies have confirmed that Terminalia bellirica has hepatoprotective, hypoglycemic, lipid-regulating, and antimicrobial activities. Commonly used compound preparations include Basangmu Butter Pills, Renqing Mangjue, and Twenty-Five Flavor Turquoise Pills.

[0003] Currently, the varieties of Terminalia chebula (including Terminalia chebula and Terminalia velutipes) and Terminalia chebula in the market are quite chaotic and cannot be traced. Judging from the origin of the commercially available samples collected, the domestic samples mainly come from Guangdong, Guangxi, Yunnan and Fujian, while the origin of imported samples is less clear.

[0004] The quality of traditional Chinese medicine (TCM) reflects and characterizes its efficacy and safety, serving as a crucial basis for controlling the effectiveness of TCM clinical use and prepared TCM products. However, by comparing the standards for Terminalia chebula and Terminalia chebula recorded in the 2020 edition of the Chinese Pharmacopoeia with those in major international pharmacopoeias, and further comparing them with the processing standards of major provincial-level Chinese medicine preparations, the results show that Terminalia chebula standards are currently included in major international pharmacopoeias such as the Chinese Pharmacopoeia, USP-NF, British Pharmacopoeia, Indian Pharmacopoeia, and Korean Pharmacopoeia, and are also included in the processing standards of various provincial-level Chinese medicine preparations. Terminalia chebula is included in the British Pharmacopoeia and Indian Pharmacopoeia, and its processing standards are included in four provincial-level Chinese medicine preparations. Terminalia chebula is included in the Chinese Pharmacopoeia and various provincial-level processing standards, but is not recorded in major international pharmacopoeias. The standards for Terminalia chebula, Terminalia chebula fruit, and Terminalia chebula fruit were included in the Chinese Pharmacopoeia relatively early and have been gradually improved during the revision of each edition. However, their testing standards are lower than those of major foreign pharmacopoeias. Current research on Terminalia chebula, Terminalia chebula fruit, and Terminalia chebula fruit mainly focuses on the analysis of their pharmacodynamic material basis and pharmacological effects. However, research on the original Terminalia chebula fruit is scarce, and studies on the chemical components and corresponding biological effects of Terminalia chebula fruit and Terminalia chebula fruit are also limited. Furthermore, due to the large amount of phenolic acids and tannins in Terminalia chebula, reports indicate that its aqueous extract can cause elevated serum AST and ALT levels in mice with acute and chronic Terminalia chebula-induced liver injury models, resulting in significant liver damage. Therefore, establishing multi-index component determination methods for Terminalia chebula, Terminalia chebula fruit, and Terminalia chebula fruit is particularly important. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining five components in Combretaceae medicinal materials based on a single-test-multiple-evaluation method, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for determining five components in Combretaceae medicinal materials based on a single-test, multi-evaluation method. The method includes the following steps:

[0008] 1) Preparation of reference solution: Prepare a mixed reference solution; the reference includes one or more of chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloglucoside, 1,2,3,4,6-O-pentagalloglucoside and ellagic acid.

[0009] 2) Preparation of test solution: Take the medicinal material of the Combretaceae family, add solvent for extraction, then add solvent to make up the weight loss, shake well, filter, and obtain the test solution; the extraction method includes cold soaking, ultrasonic extraction or reflux extraction;

[0010] 3) The reference solution was diluted separately and then injected into the test solution into a high-performance liquid chromatograph for chromatographic detection. The chromatographic conditions were: C1... 18 The chromatographic column used methanol as mobile phase A and acid-water solution as mobile phase B, with the gradient elution program shown in the table below:

[0011] 0-30 24→27 76→73 30-50 27→42 73→58 50-65 42→90 58→10

[0012] The flow rate is 0.6-1.5 mL / min; the column temperature is 30-40℃; the detection wavelength is 190-400 nm; and the injection volume is 3-10 μL.

[0013] 4) Based on the test results, obtain information about the Combretaceae medicinal material; the information includes component information and / or content information;

[0014] The medicinal materials of the Combretaceae family include one or more of the following: Terminalia chebula, Terminalia chebula var. hainanensis, and Terminalia chebula.

[0015] Preferably, the method further includes using chebulic acid as an internal reference to calculate the relative retention value and relative correction factor of the target compound; the target compound includes one or more of chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloglucoside, 1,2,3,4,6-O-pentagalloglucoside, and ellagic acid.

[0016] More preferably, the flow rate is 0.8-1.2 mL / min; the column temperature is 32-37℃; the detection wavelength is 260-290 nm; and the injection volume is 5-10 μL.

[0017] More preferably, the flow rate is 1.0 mL / min; the column temperature is 35 °C; the detection wavelength is 275 nm; and the injection volume is approximately 10 μL.

[0018] Preferably, the solvent is methanol with a volume percentage of 40-90%;

[0019] And / or, the power of the ultrasound is 100-500W, the frequency is 20-100kHz, and the duration is 5-30min;

[0020] More preferably, the solvent is methanol with a volume percentage of 50-80%;

[0021] And / or, the power of the ultrasound is 200-400W, the frequency is 30-80kHz, and the duration is 10-25min;

[0022] More preferably, the solvent is methanol with a volume percentage of 70%;

[0023] The ultrasound had a power of 350W, a frequency of 53kHz, and a duration of 15min.

[0024] Preferably, the C 18 The chromatographic column includes Phenomenex Luna C10. 18 Thermo Acclaim C 18 Or Welch Ultimate XB C 18 .

[0025] Preferably, the acid aqueous solution is a 0.1%-0.3% acid aqueous solution;

[0026] The acidic aqueous solution includes an aqueous solution of phosphoric acid.

[0027] More preferably, the acidic aqueous solution is a 0.2% phosphoric acid aqueous solution.

[0028] Preferably, the resolution R of each chromatographic peak is greater than 1.5, and the tailing factor T is 0.95-1.05.

[0029] Preferably, when chebulic acid is used as an internal reference, the target compound includes chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloylglucose, 1,2,3,4,6-O-pentagalloylglucose, and ellagic acid.

[0030] Preferably, when chebulic acid is used as an internal reference, the relative retention values ​​are used to locate the chromatographic peaks of the target compound; the relative retention value of chebulic acid is 0.52±5%; the relative retention value of 1,2,3,6-O-tetragalloylglucose is 0.47±5%; the relative retention value of 1,2,3,4,6-O-pentagalloylglucose is 0.90±5%; and the relative retention value of ellagic acid is 1.07±5%.

[0031] When chebulic acid is used as an internal reference, the relative correction factor is used to calculate the content of the target compound; the relative correction factor for chebulic acid is 1.34±5%; the relative correction factor for 1,2,3,6-O-tetragalloylglucose is 0.81±5%; the relative correction factor for 1,2,3,4,6-O-pentagalloylglucose is 0.78±5%; and the relative correction factor for ellagic acid is 0.83±5%.

[0032] More preferably, when chebulic acid is used as an internal reference, the relative retention values ​​are used to locate the chromatographic peaks of the target compound; the relative retention value of chebulic acid is 0.52; the relative retention value of 1,2,3,6-O-tetragalloyl glucose is 0.47; the relative retention value of 1,2,3,4,6-O-pentagalloyl glucose is 0.90; and the relative retention value of ellagic acid is 1.07.

[0033] When chebulic acid is used as an internal reference, the relative correction factor is used to calculate the content of the target compound; the relative correction factor for chebulic acid is 1.34; the relative correction factor for 1,2,3,6-O-tetragalloylglucose is 0.81; the relative correction factor for 1,2,3,4,6-O-pentagalloylglucose is 0.78; and the relative correction factor for ellagic acid is 0.83.

[0034] This invention provides the application of the above-described method in detecting the content of chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloyl glucose, 1,2,3,4,6-O-pentagalloyl glucose and ellagic acid in Combretaceae medicinal materials, wherein the Combretaceae plants include one or more of Terminalia chebula, Terminalia chebula and Terminalia chebula.

[0035] This invention provides the application of the above-described method in evaluating the quality of Combretaceae medicinal materials, wherein the Combretaceae plants include one or more of Terminalia chebula, Terminalia chebula var. chinensis, and Terminalia chebula.

[0036] This invention provides the application of the above-described method in evaluating the quality of Combretaceae medicinal materials, evaluating the authenticity of Combretaceae medicinal materials, or in the quality control of Combretaceae medicinal materials, wherein the Combretaceae medicinal materials include one or more of Terminalia chebula, Terminalia chebula var. hainanensis, and Terminalia chebula.

[0037] The present invention discloses the following technical effects:

[0038] 1. This invention establishes a method for the simultaneous quantitative detection of five components in Combretaceae medicinal materials based on a single-measurement-multiple-evaluation method. By simultaneously determining the content of the five component indicators using this method, only chebulic acid is needed as an internal reference. The specific contents of chebulic acid, 1,2,3,6-O-tetragalloylglucose, 1,2,3,4,6-O-pentagalloylglucose, and ellagic acid can be calculated through relative correction factors. This method has the advantages of simple operation, high sensitivity, accurate determination, and strong applicability.

[0039] 2. This invention uses the traditional internal standard method and the one-measure-multiple-evaluation method to compare and measure the differences in the content of five components. The results prove that this invention meets the requirements of methodological validation and is feasible. Compared with existing methods based on internal standards, this invention not only overcomes the shortcomings of some reference standards in multi-index quantitative analysis, such as difficulty in preparation, high price, difficulty in mass supply, and even lack of commercially available products, but also enables the simultaneous determination of multiple index components of traditional Chinese medicine at low cost and high efficiency, comprehensively and objectively reflecting the intrinsic quality of traditional Chinese medicine. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 The HPLC chromatograms are of the mixed reference standard (A), Terminalia chebula herb (No.: HZ-4) (B), and Terminalia chebula herb (No.: HP-1-1) (C); where 1 represents TeGG; 2 represents chebulic acid; 3 represents PGG; 4 represents chebulic acid; and 5 represents ellagic acid.

[0042] Figure 2 The HPLC chromatograms are of mixed reference standard (A), Terminalia chebula slices (No.: HZ-9) (B), and Terminalia chebula slices (No.: HSZ-2-2) (C); where 1 represents TeGG; 2 represents chebulic acid; 3 represents PGG; 4 represents chebulic acid; and 5 represents ellagic acid.

[0043] Figure 3 The HPLC chromatograms are of the mixed reference standard (A), Terminalia chebula pulp slices (No.: HZR-41) (B), and Terminalia chebula pulp slices (No.: HZR-46) (C); where 1 represents TeGG; 2 represents chebulic acid; 3 represents PGG; 4 represents chebulic acid; and 5 represents ellagic acid.

[0044] Figure 4 The HPLC chromatograms are of mixed reference standard (A) and Terminalia chebula medicinal material (No.: MHZ-14) (B); where 1 is TeGG; 2 is chebulic acid; 3 is PGG; 4 is chebulic acid; and 5 is ellagic acid.

[0045] Figure 5 The HPLC chromatograms are of mixed reference standard (A) and Terminalia chebula medicinal material (No.: XQG-4) (B); where 1 represents TeGG; 2 represents chebulic acid; 3 represents PGG; 4 represents chebulic acid; and 5 represents ellagic acid.

[0046] Figure 6 This is a full-wavelength scan of the five components to be tested. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] Sample collection and preparation:

[0053] This invention collected or field-collected 56 batches of Terminalia chebula medicinal materials, including 33 batches of commercially available Terminalia chebula samples (31 batches of samples with T. chebula as the source and 2 batches of samples with T. chebula var. tomentella as the source), and 23 batches of field-collected Terminalia chebula samples (3 batches of samples with T. chebula as the source and 20 batches of samples with T. chebula var. tomentella as the source), and 20 batches of field-collected Terminalia chebula samples (see Table 2 for details). The above-mentioned field-collected Terminalia chebula samples were harvested and processed according to the methods in the 2020 edition of the Chinese Pharmacopoeia. The harvesting and processing methods were as follows: harvesting when the fruit is ripe in autumn and winter, removing impurities, and sun-drying.

[0054] This invention collected or prepared 37 batches of Terminalia chebula slices (17 batches of samples with T. chebula as the source, including 13 commercially available batches and 4 self-prepared batches; 20 batches of samples with T. chebula var. tomentella as the source, all of which were self-prepared due to the near absence of commercially available cleaned Terminalia chebula slices with villi), and detailed sample information is shown in Table 3; it also collected or prepared 32 batches of Terminalia chebula pulp slices (16 batches of samples with T. chebula as the source, including 14 commercially available batches and 2 self-prepared batches; 16 batches of samples with T. chebula var. tomentella as the source, including 4 commercially available batches and 12 self-prepared batches), and detailed sample information is shown in Table 4; the above self-prepared Terminalia chebula slices were processed according to the Terminalia chebula processing method under the 2020 edition of the Chinese Pharmacopoeia: removing impurities, washing, and drying. They were then crushed before use. The samples of Terminalia chebula pulp were processed according to the method for processing Terminalia chebula pulp under the

Processing

[0055] This invention collected 32 batches of commercially available Terminalia chebula samples, and the specific sample information is shown in Table 5; it also collected 15 batches of commercially available Terminalia chebula medicinal materials, and the specific sample information is shown in Table 6.

[0056] The aforementioned 56 batches of Terminalia chebula medicinal material samples, 37 batches of Terminalia chebula sliced ​​medicinal material samples, and 32 batches of Terminalia chebula pulp samples were identified by Researcher Guo Baolin of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, as dried mature fruits of Terminalia chebula Retz. or Terminalia chebula Retz. var. tomentella Kurt., plants of the Combretaceae family; 15 batches of Terminalia bellirica medicinal material were identified by Researcher Guo Baolin as dried mature fruits of Terminalia bellirica (Gaertn.) Roxb., a plant of the Combretaceae family; and 32 batches of Terminalia chebula fruit medicinal material were identified by Researcher Guo Baolin as dried immature fruits of Terminalia chebula Retz., a plant of the Combretaceae family.

[0057] Table 1 Information on Commercially Available Terminalia chebula Herbs

[0058]

[0059]

[0060] Table 2 Information on samples of Terminalia chebula medicinal materials collected on-site

[0061]

[0062]

[0063] Table 3. Information on processed Terminalia chebula slices (cleaned)

[0064]

[0065]

[0066] Table 4. Sample Information of Terminalia chebula Meat Slices

[0067]

[0068]

[0069] Note: "-" indicates unknown.

[0070] Table 5 Information on Terminalia chebula Samples

[0071]

[0072]

[0073] Table 6. Information on Terminalia chebula samples

[0074] MHZ-1 Traditional Chinese Medicine Trading Market, Qiaocheng District, Bozhou City, Anhui Province India 2023.04.17 MHZ-2 Chengdu, Sichuan Province India 2023.04.17 MHZ-3 Anhui Bozhou City Qiaocheng District Fuzhiyuan Agricultural Development Co., Ltd. Guangxi 2023.07.14 MHZ-4 Traditional Chinese Medicine Wholesale Market, Qiaocheng District, Bozhou City, Anhui Province Anhui 2023.07.14 MHZ-5 Qiaocheng District, Bozhou City, Anhui Province Sichuan 2023.07.14 MHZ-6 Anguo Traditional Chinese Medicine Market, Baoding City, Hebei Province Henan 2023.07.14 MHZ-7 Shennong Valley Traditional Chinese Medicine E-commerce City, Qiaocheng District, Bozhou City, Anhui Province Tibet 2023.07.14 MHZ-8 Shennong Valley Traditional Chinese Medicine E-commerce City, Qiaocheng District, Bozhou City, Anhui Province Tibet 2023.07.14 MHZ-9 Kangmei Medicinal Herbs Market, Qiaocheng District, Bozhou City, Anhui Province Tibet 2023.07.14 MHZ-10 Qiaocheng District, Bozhou City, Anhui Province India 2023.07.18 MHZ-11 Qiaocheng District, Bozhou City, Anhui Province Tibet 2023.07.18 MHZ-12 Qiaocheng District, Bozhou City, Anhui Province Tibet 2023.07.18 MHZ-13 Qiaocheng District, Bozhou City, Anhui Province Tibet 2023.07.18 MHZ-14 Qiaocheng District, Bozhou City, Anhui Province Yunnan 2023.07.18 MHZ-15 Qiaocheng District, Bozhou City, Anhui Province Guangdong 2023.07.20

[0075] Example 1

[0076] 1. Instruments and Materials

[0077] The system included: WatersAcquity UPLC / Xevo G2-XS Q-TOF ultra-high performance liquid chromatography / high resolution mass spectrometry analyzer (Waters Corporation, USA); Shimadzu LC-20AT high performance liquid chromatograph (Shimadzu Corporation, Japan); Watersalliance e2695 high performance liquid chromatograph (Waters Corporation, USA); Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); FW100 high-speed grinder (Tianjin Tester Instrument Co., Ltd.); SK7210HP CNC ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); ME104 Mettler 0.001 g electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); and METTLER TOLEDO Mettler 0.001 g electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.). Chromatographic column: ThermoAcclaim C 18 Chromatographic column (4.6 × 250 mm, 5 μm), Phenomenex LunaC 18 Chromatographic column (4.6 × 250 mm, 5 μm), Welch Ultimate XB C 18 Chromatographic column (4.6×250mm, 5μm).

[0078] The reference standards chebulagic acid (batch number: HR8096W4, purity 98%), chebulinic acid (batch number: HR8095W5, purity 98%), 1,2,3,6-O-tetragalloglucopyranoside (TeGG, batch number: HR9213W2, purity 98%), 1,2,3,4,6-O-pentagalloglucopyranoside (PGG, batch number: HR2331W17, purity ≥98%), and ellagic acid (batch number: HR8331W11, purity 98%) were all purchased from Baoji Chenguang Biotechnology Co., Ltd. Methanol and acetonitrile were chromatographic grade, sourced from Fisher Scientific, USA; phosphoric acid was chromatographic grade, sourced from Beijing Mairuida Technology Co., Ltd.; methanol was analytical grade; and water was Wahaha purified water.

[0079] 2. Establishment of chromatographic conditions

[0080] Column: Thermo Acclaim tm C 18 Chromatographic column (4.6 × 250 mm, 5 μm); mobile phase: gradient elution of methanol (A) and 0.2% phosphoric acid aqueous solution (B), 0–30 min, 24%–27% A; 30–50 min, 27%–42% A; 50–65 min, 42%–90% A; column temperature 35℃, flow rate 1.0 mL·min -1 The detection wavelength was 275 nm, and the injection volume was 10 μL. Under the current chromatographic conditions, all five analytes achieved baseline separation, with peak resolution R > 1.5 and tailing factor T ≈ 1.00, indicating good separation performance. The HPLC chromatograms of the mixed reference standard (mixed reference standard solution, preparation method detailed in step 3) and the test solutions of Terminalia chebula and Terminalia velutipes medicinal materials (preparation method detailed in step 4, the final determined method) are shown below. Figure 1 The HPLC chromatograms of the mixed reference standard and Terminalia chebula and Terminalia chebula slices are shown in [reference image]. Figure 2 The HPLC chromatograms of the mixed reference standard (mixed reference standard solution, preparation method detailed in step 3) and the test solution of Terminalia chebula pulp and Terminalia chebula pulp slices (preparation method detailed in step 4, the final determined method) are shown below. Figure 3 The HPLC chromatograms of the mixed reference standard (mixed reference standard solution, preparation method detailed in step 3) and the Terminalia chebula medicinal material test solution (preparation method detailed in step 4, the final method determined) are shown below. Figure 4 The HPLC chromatograms of the mixed reference standard and Terminalia chebula medicinal material are shown below. Figure 5 .

[0081] Selection of UV detection wavelength: This invention performed a full-wavelength scan of five components in the Terminalia chebula test sample solution (preparation method detailed in step 4, final determination method) at wavelengths of 190-400 nm. For example... Figure 6 As shown, TeGG (1,2,3,6-O-tetragalloglucoside), chebulic acid, PGG (1,2,3,4,6-O-pentagalloglucoside), and chebulic acid have maximum absorption at wavelengths of 277 nm, 275 nm, 279 nm, and 278 nm, respectively, while ellagic acid has maximum absorption at 252 nm. Considering the maximum absorption wavelength and absorption range of the above five components, the detection wavelength selected in this experiment is 275 nm.

[0082] 3. Preparation of reference solution

[0083] Accurately weigh 4.86, 6.04, and 3.72 mg of TeGG, PGG, and chebulic acid reference standards, respectively, and place them in 5 mL volumetric flasks. Add 70% methanol solution to prepare mass concentrations of 972, 1208, and 744 μg·mL, respectively. -1 Reference stock solution;

[0084] Separately, accurately weigh 2.50 mg of ellagic acid reference standard, place it in a 25 mL volumetric flask, add an appropriate amount of dimethyl sulfoxide (DMSO) to aid dissolution, and then add 70% methanol solution to prepare a solution with a mass concentration of 100 μg·mL. -1 Ellagic acid stock solution.

[0085] Accurately weigh 6.51 mg of chebulic acid reference standard and place it in a 10 mL volumetric flask. Accurately measure 2 mL of the above-mentioned TeGG stock solution, 2 mL of PGG stock solution, and 3 mL of chebulic acid stock solution into the same 10 mL volumetric flask, dissolve, and dilute to volume. The resulting concentrations of chebulic acid, TeGG, PGG, and chebulic acid are 651, 194.4, 241.6, and 223.2 μg·mL, respectively. -1 1# Mixed reference solution.

[0086] Dilute the No. 1 mixed reference solution by 2 times to obtain the No. 2 mixed reference solution; accurately measure 2 mL of the No. 1 mixed reference solution and 4 mL of ellagic acid stock solution into the same volumetric flask (10 mL), and add 70% methanol solution to prepare the No. 3 mixed reference solution; accurately measure 2 mL each of the No. 2 mixed reference solution and ellagic acid stock solution into the same volumetric flask (10 mL), and add 70% methanol solution to prepare the No. 4 mixed reference solution; dilute the No. 3 mixed reference solution by 5 times to obtain the No. 5 mixed reference solution; dilute the No. 4 mixed reference solution by 5 times to obtain the No. 6 mixed reference solution; dilute the No. 6 mixed reference solution by 2 times to obtain the No. 7 mixed reference solution.

[0087] In summary, the concentrations of TeGG in the prepared mixed control sample solutions were: 194.4, 97.20, 38.88, 19.44, 7.776, 3.888, and 1.944 μg·mL, respectively. -1The concentration series of chebulic acid were: 651.0, 325.5, 130.2, 65.10, 26.04, 13.02, and 6.510 μg·mL. -1 The PGG concentration series were: 241.6, 120.8, 48.32, 24.16, 9.664, 4.832, and 2.416 μg·mL. -1 The concentration series of chebulic acid were 223.2, 111.6, 44.64, 22.32, 8.928, 4.464, and 2.232 μg·mL, respectively. -1 The concentration series of ellagic acid are as follows: 100.0 (ellagic acid mother liquor, also known as ellagic acid 1#), 40.00 (ellagic acid 2#), 20.00 (ellagic acid 3#), 8.00 (ellagic acid 4#), 4.00 (ellagic acid 5#), and 2.00 (ellagic acid 6#) μg·mL. -1 .

[0088] Note: Ellagic acid reference standard is poorly soluble in methanol and methanol in varying proportions, requiring the addition of DMSO for dissolution. Even after adding DMSO, its concentration cannot reach 200 μg / mL. -1 Therefore, the injection volume was doubled (20 μL) as the first concentration point of the standard curve.

[0089] 4. Preparation of the test solution

[0090] Through investigation of different extraction methods (cold soaking, reflux, or ultrasound), different extraction times (5, 15, 30, 45, 60 min), different extraction solvents (50% methanol, 60% methanol, 70% methanol, 80% methanol, "%" is volume percentage), and solvent dosage (60, 80, 100, 120 times), the preparation method of the test solution was determined as follows: Take about 0.25 g of medicinal material powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 70% methanol solution, weigh it, sonicate it (power 350 W, frequency 53 kHz) for 15 min, cool it, weigh it again, replenish the lost weight with 70% methanol solution, shake well, filter it, take the filtrate (plant source Terminalia chebula) or the filtrate (plant source Terminalia chebula, Terminalia chebula, and Terminalia chebula), dilute it 5 times, and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0091] 5. Methodological Investigation

[0092] 5.1 Linear range, detection limit, and quantitation limit

[0093] Accurately pipette 10 μL of the mixed reference solution (1-7#) from step 3, and 10 μL and 20 μL of the ellagic acid stock solution, respectively. Inject these solutions under the chromatographic conditions described in step 2 to determine the peak areas of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid. Calculate the peak areas using mass concentration X (μg·mL⁻¹). -1 Linear regression analysis (passing through the origin) was performed on the peak area Y to obtain the regression equation, correlation coefficient (r), and linear range for each component. Simultaneously, the mixed reference standard #7 was serially diluted, injected, and analyzed to determine the peak area. The limit of detection (LOD) for each component was set at a signal-to-noise ratio (S / N) of 3, and the limit of quantitation (LOQ) was set at an S / N of 10. The results are shown in Table 7. The results indicate that the five components exhibit good linearity and sensitivity over a relatively wide concentration range (50-fold or 100-fold).

[0094] Table 75 shows the regression equations, linear ranges, limits of detection, and limits of quantitation for each component.

[0095]

[0096] 5.2 Precision test

[0097] The test solution of Terminalia chebula was prepared according to the method determined in step 4. Six consecutive injections were performed, and the peak area was measured. The RSD was calculated, and the results are shown in Table 8. The results show that the RSDs of the peak areas of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid were 1.45%, 0.13%, 1.68%, 0.12%, and 0.91%, respectively. These results indicate that the instrument has good precision.

[0098] Table 8. Results of the instrument precision test (n=6)

[0099]

[0100] 5.3 Stability Test

[0101] 5.3.1 Stability of the reference solution

[0102] Take the No. 3 mixed reference solution from step 3 and analyze it at 0, 6, 12, 24, 36, and 48 hours after preparation, according to the chromatographic conditions in step 2. Measure the peak area and calculate the RSD. The results are shown in Table 9. The results show that the RSDs of the peak areas of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid are 0.57%, 4.30%, 2.45%, 0.67%, and 0.10%, respectively. This indicates that the reference solution has good stability within 48 hours.

[0103] Table 9. Results of the stability study of the mixed reference standards (n=6)

[0104]

[0105] 5.3.2 Stability of the test solution

[0106] The test solution of Terminalia chebula was prepared according to the method determined in step 4. Chromatographic analysis was performed at 0, 6, 12, 17, 36, and 48 hours after preparation, following the chromatographic conditions in step 2. Peak areas were measured, and RSDs were calculated. The results are shown in Table 10. The results showed that the RSDs of the peak areas of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid were 2.57%, 3.81%, 0.90%, 0.71%, and 0.28%, respectively. This indicates that the test solution exhibited good stability within 48 hours.

[0107] Table 10 Results of the stability study of the test solutions (n=6)

[0108]

[0109] 5.4 Repeatability Test

[0110] Accurately weigh approximately 0.25 g of Terminalia chebula powder, prepare six parallel aliquots, and administer the test solution according to the method determined in step 4. Inject the solution under the chromatographic conditions specified in step 2, determine the peak area, and calculate the RSD. The results are shown in Table 11. The results show that the RSDs of the peak areas of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid are 2.01%, 2.21%, 2.30%, 1.92%, and 1.08%, respectively, indicating that the method has good repeatability.

[0111] Table 11 Results of Repeatability Testing (n=6)

[0112]

[0113] 5.5 Recovery rate

[0114] Approximately 25 mg of Terminalia chebula powder with known component content was accurately weighed using a 1 / 100,000 balance. Six parallel aliquots were prepared, and the sample was added to the reference solution at a ratio of approximately 1:1 (sample to reference solution in μg). The test solution was prepared according to the method determined in step 4, and the samples were injected for analysis. The recoveries and RSDs of each component were determined and calculated. The results are shown in Table 12. The results showed that the average recoveries of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid were 98.56%, 99.14%, 95.14%, 99.62%, and 99.05%, respectively; the RSDs (n=6) were 1.40%, 2.45%, 2.10%, 1.16%, and 2.47%, respectively, indicating that the method has good accuracy.

[0115] Table 1. Results of recovery rate study for 125 components (n=6)

[0116]

[0117] 6. Establishment of relative correction factors

[0118] 6.1 Calculation of Relative Correction Factor

[0119] The reference solution (mixed reference solution) was prepared using the slope method as described in step 3. It was then injected and analyzed under the chromatographic conditions described in step 2. The peak areas of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid were determined, and the concentrations were expressed as X (mg·L⁻¹). -1 Linear regression was performed on the peak area Y to obtain the regression equations, slopes, and correlation coefficients (r) for each component. Terminalia chebulic acid was used as an internal reference (s), and the results were obtained according to formula f. s / x =K s / K x Calculate the relative correction factors (RCF) for TeGG, chebulic acid, PGG, and ellagic acid, where f s / x Kx is the RCF of component x relative to internal reference s, Ks is the slope of the standard curve of internal reference s, and Kx is the slope of the standard curve of component x. The results are shown in Table 13.

[0120] Using a multi-point method, based on the concentrations and measured peak areas of the six mixed standard solutions (mixed reference solutions) in step 3, the relative correction factors for TeGG, chebulic acid, PGG, and ellagic acid were calculated according to the following formula:

[0121]

[0122] In the formula: A s A represents the peak area of ​​the internal reference substance s. s A represents the concentration or mass of the internal reference substance S. k C represents the peak area of ​​the component to be measured. k The concentration or mass of the component to be tested is shown in Table 14.

[0123] Table 13 Results of relative correction factor determination using the slope method (n=6)

[0124]

[0125] Table 14 Results of Multi-Point Method for Determining Relative Correction Factor

[0126]

[0127] When calculating RCFs using the slope method, a larger correlation coefficient indicates a more accurate calculation. Similarly, when calculating RCFs using the multi-point method, a smaller RSD indicates a more accurate calculation. Tables 13 and 14 show that the correlation coefficient r of the regression equations for the four components is ≥0.999, indicating a good linear relationship. However, the RSD of the RCFs for TeGG, PGG, and ellagic acid calculated using the multi-point method is >5%, indicating that this method is not suitable. Therefore, the slope method is used to establish relative correction factors for TeGG, chebulic acid, PGG, and ellagic acid.

[0128] 6.2 Repeatability test of relative correction factor

[0129] Prepare the mixed reference solutions 1#-6# and ellagic acid 1# and ellagic acid 2# according to the method in step 3. The average value of one test is the result of the first part. Six parallel tests were performed (i.e., six sets of mixed reference solutions 1#-6# and ellagic acid 1# and ellagic acid 2# were prepared). The RCFs, mean, and RSD of TeGG, chebulic acid, PGG, and ellagic acid were calculated using the slope method. The results are shown in Table 15. The results show: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​were 0.784, 1.310, 0.794 and 0.847, respectively, and the RSDs were 2.85%, 2.45%, 1.83 and 2.31%, respectively, indicating that the repeatability of the relative correction factors was good.

[0130] Table 15. Repeatability test results of relative correction factors (using chebulic acid as an internal reference) (n=6)

[0131]

[0132]

[0133] Note: f 诃子酸 / TeGG f is the relative correction factor for TeGG; 诃子酸 / 诃黎勒酸 The relative correction factor for chebulic acid; f 诃子酸 / PGG f is the relative correction factor for PGG; 诃子酸 / 鞣花酸 This is the relative correction factor for ellagic acid; the same applies below.

[0134] 6.3 Robustness test of relative correction factor

[0135] 6.3.1 Effect of different HPLC instruments on relative correction factor

[0136] Welch Ultimate XB C 18Chromatographic columns were used on three HPLC instruments: a Shimadzu LC-20A, an Agilent 1260, and a Waters e2695, respectively, to analyze f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 16. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​on the three HPLC instruments were 0.799, 1.328, 0.771, and 0.825, respectively, with RSDs of 2.09%, 3.60%, 2.82%, and 1.06%, all <4.00%, indicating that the Welch Ultimate XB C... 18 When using chromatographic columns, the relative correction factor exhibits good durability across different HPLC instruments.

[0137] Table 16 Same chromatographic column (Welch Ultimate XB C) 18 The effect of different HPLC instruments on the relative correction factor (n=3)

[0138]

[0139] Using Thermo Acclaim C 18 Chromatographic columns were used on three HPLC instruments: a Shimadzu LC-20A, an Agilent 1260, and a Waters e2695, respectively, to analyze f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 17. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​on the three HPLC instruments were 0.801, 1.377, 0.779, and 0.820, respectively, with RSDs of 0.11%, 5.69%, 2.97%, and 2.31%, all <6.00%, indicating that the Thermo Acclaim C 18 When using chromatographic columns, the relative correction factor exhibits good durability across different HPLC instruments.

[0140] Table 17 Same chromatographic column (Thermo Acclaim C) 18The effect of different HPLC instruments on the relative correction factor (n=3)

[0141]

[0142] Phenomenex Luna C 18 Chromatographic columns were used on three HPLC instruments: a Shimadzu LC-20A, an Agilent 1260, and a Waters e2695, respectively, to analyze f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 18. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​on the three HPLC instruments were 0.811, 1.328, 0.787, and 0.808, respectively, with RSDs of 1.13%, 2.01%, 2.68%, and 0.40%, all <3.00%, indicating that the Phenomenex Luna C4000 HPLC system was effective. 18 When using a chromatographic column, the relative correction factor exhibits good robustness across different HPLC instruments.

[0143] Table 18. Same chromatographic column (Phenomenex Luna C) 18 The effect of different HPLC instruments on the relative correction factor (n=3)

[0144]

[0145] 6.3.2 Effect of different chromatographic columns on relative correction factors

[0146] A Shimadzu LC-20A HPLC system and a Phenomenex Luna C were used. 18 Thermo Acclaim C 18 Welch Ultimate XB C 18 Three types of chromatographic columns were used to treat f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 19. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸The mean values ​​on the three columns were 0.806, 1.330, 0.793 and 0.825, respectively, and the RSDs were 1.16%, 2.50%, 1.46% and 1.83%, respectively, all <3.00%, indicating that the relative correction factor on the Shimadzu LC-20A HPLC instrument has good robustness to different columns.

[0147] Table 19. Effect of different chromatographic columns on the relative correction factor for the same HPLC instrument (Shimadzu LC-20A) (n=3)

[0148]

[0149] Waters e2695 HPLC system and Phenomenex Luna C were used. 18 Thermo Acclaim C 18 Welch Ultimate XB C 18 Three types of chromatographic columns were used to treat f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 20. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​on the three columns were 0.806, 1.331, 0.789 and 0.808, respectively, with RSDs of 1.07%, 4.35%, 0.91% and 0.86%, respectively, all <5.00%, indicating that the relative correction factor on the Waters e2695 HPLC instrument has good robustness to different columns.

[0150] Table 20. Effect of different chromatographic columns on the relative correction factor for the same HPLC instrument (Waters e2695) (n=3)

[0151]

[0152] An Agilent 1260 HPLC system and a Phenomenex Luna C system were used. 18 Thermo Acclaim C 18 Welch Ultimate XB C 18 Three types of chromatographic columns were used to treat f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 21. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​on the three columns were 0.800, 1.407, 0.754 and 0.819, respectively, with RSDs of 2.07%, 6.51%, 1.25% and 1.63%, respectively, all <7.00%, indicating that the relative correction factor has good robustness to different columns on the Agilent 1260-HPLC instrument.

[0153] Table 21 Effect of different columns on relative correction factors for the same HPLC instrument (Agilent 1260) (n=3)

[0154]

[0155] 6.3.3 Effect of different flow velocities on relative correction factor

[0156] The Shimadzu LC-20A HPLC system and Thermo Acclaim C were used. 18 The chromatographic column was used at different flow rates (0.90, 1.00, 1.10 mL·min). -1 Under the condition of f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 22. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​under different flow rates were 0.744, 1.329, 0.785 and 0.830, respectively, and the RSDs were 0.86%, 2.84%, 1.19% and 0.59%, respectively, all <3.00%, indicating that the relative correction factor has good durability under different flow rates.

[0157] Table 22 Effect of different flow velocities on the relative correction factor (n=3)

[0158]

[0159] 6.3.4 Effect of different column temperatures on relative correction factor

[0160] The Shimadzu LC-20A HPLC system and Thermo Acclaim C were used. 18The chromatographic column was subjected to different column temperatures (33, 35, 37℃) to test f. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The results are shown in Table 23. The results show that: f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​under different column temperatures were 0.764, 1.334, 0.770 and 0.833, respectively, and the RSDs were 1.06%, 3.17%, 0.56% and 0.38%, respectively, indicating that the relative correction factor has good durability under different column temperatures.

[0161] Table 23 Effect of different column temperatures on relative correction factors (n=3)

[0162]

[0163]

[0164] 6.4 Intermediate precision of relative correction factors

[0165] The f samples were analyzed by different personnel within the organization on different working days using HPLC instruments in three different laboratories. 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The determination was performed using mixed reference solutions 1#-6# prepared according to the method in step 3, as well as ellagic acid 1# and ellagic acid 2#. The mean and RSD were calculated. Results f 诃子酸 / TeGG f 诃子酸 / 诃黎勒酸 f 诃子酸 / PGG and f 诃子酸 / 鞣花酸 The mean values ​​were 0.81, 1.34, 0.78 and 0.83, respectively; the RSDs (n=3) were 1.53%, 2.78%, 3.44% and 3.00%, respectively, indicating that the intermediate precision of the relative correction factor was good. The results are shown in Table 24.

[0166] Table 24. Reproducibility test results of the relative correction factor (with chebulic acid as an internal reference) (n=3)

[0167]

[0168] 6.5 Determination of Relative Correction Factor

[0169] Finally, based on the intermediate precision test results of the relative correction factors, the relative correction factors (RCFs) for TeGG, chebulic acid, PGG, and ellagic acid were determined to be fi, fj ...诃子酸 / TeGG =0.81, f 诃子酸 / 诃黎勒酸 =1.34, f 诃子酸 / PGG =0.78, and f 诃子酸 / 鞣花酸 =0.83.

[0170] 7. Localization of chromatographic peaks of the analyte

[0171] Phenomenex C 18 Thermo Acclaim C 18 Welch Ultimate XB C 18 The retention times (t) of five components were determined using three HPLC instruments: a Shimadzu LC-20A, an Agilent 1260, and a Waters e2695. R Using chebulic acid as an internal reference (S), according to formula r x / s =t Rx / t Rs and r x-s =t Rx -t Rs Calculate the relative retention times and retention time differences of TeGG, chebulic acid, PGG, and ellagic acid, where r is the relative retention time and retention time difference. x / s r x-s t represents the relative retention time and retention time difference of component x relative to internal reference s. Rx Let t be the retention time of component x. Rs The retention times of the internal reference s are shown in Tables 25 and 26. The RSDs of the relative retention values ​​of the four analytes ranged from 0.59% to 4.35%, and the RSDs of the retention time differences ranged from -5.60% to 10.44%. These results indicate that the relative retention value method is more accurate in locating chromatographic peaks compared to the retention time difference method.

[0172] Therefore, when chebulic acid was used as an internal control, the relative retention values ​​of TeGG, chebulic acid, PGG, and ellagic acid were finally determined to be: r TeGG / 诃子酸 =0.47, r 诃黎勒酸 / 诃子酸 =0.52, r PGG / 诃子酸 =0.90, r 鞣花酸 / 诃子酸 =1.07.

[0173] Table 25. Relative retention times of analytes when chebulic acid is used as an internal reference (n=9)

[0174]

[0175]

[0176] Table 26 shows the retention time differences of the analytes when chebulic acid is used as an internal reference (n=9).

[0177]

[0178] 8. Comparison of results between the one-test-multiple-evaluation method and the external standard method

[0179] The contents of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid in 12 batches of samples were determined using the External Standard Method (ESM) and the Quality Assay Method (QAMS) established in this invention, respectively. The QAMS determination was performed according to formula C. x =(A x / A s )×C s ×f s / x Perform calculations, where C x A represents the mass concentration of the analyte x in the test solution. x A represents the peak area of ​​the analyte. s C represents the peak area of ​​the reference solution of the internal control. s f represents the mass concentration of the reference solution of the internal control. s / x This represents the relative correction factor for the analyte x. The content determination results obtained from the two methods are evaluated using the relative deviation (RD). In the formula: X Q For QAMS calculated values, The average value calculated by the two methods is shown in Table 27.

[0180] The results showed that when the content of TeGG, chebulic acid, PGG and ellagic acid in the sample was determined by the one-test-multiple-evaluation method, the RD was <4%, indicating that the determination of the content of the four components TeGG, chebulic acid, PGG and ellagic acid by using chebulic acid as an internal reference was accurate and reliable.

[0181] Table 27 Comparison of the results of two methods for determining the content of five components in 12 samples.

[0182]

[0183]

[0184] Summary of 9 Content Determination Methods

[0185] Based on the above research, the relative correction factor method of "one test, multiple evaluations" was used to establish the content determination methods for five components—TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid—in Terminalia chebula medicinal materials and processed slices, Terminalia chebula medicinal materials, and Terminalia chebula medicinal materials, as shown below:

[0186] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; the chromatographic column was a Phenomenex Luna C1000. 18 Thermo Acclaim C 18 Or Welch Ultimate XB C 18 Using methanol as mobile phase A and 0.2% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed according to the specifications in Table 28; the detection wavelength was 275 nm. The theoretical plate number, calculated based on the chebulic acid peak, should not be less than 10,000.

[0187] Table 28 System Gradient

[0188] 0-30 24→27 76→73 30-50 27→42 73→58 50-65 42→90 58→10

[0189] Preparation of reference solution: Take an appropriate amount of chebulic acid reference standard, accurately weigh it, and add 70% methanol solution to prepare a solution containing 100 μg per mL.

[0190] Preparation of the test solution: Weigh approximately 0.25 g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, accurately add 20 mL of 70% methanol solution, weigh the solution, sonicate (350 W power, 53 kHz frequency) for 15 min, cool, weigh the solution again, replenish the lost weight with 70% methanol solution, shake well, filter, take the filtrate or dilute the filtrate 5 times, and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0191] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine. Using chebulic acid reference standard as a reference and its corresponding peak as the S peak, calculate the relative retention times of TeGG, chebulic acid, PGG, chebulic acid, and ellagic acid. The relative retention times should be within ±5% of the specified value. Relative retention times and correction factors are shown in Table 29.

[0192] Table 29 Relative retention times and correction factors for different components

[0193]

[0194]

[0195] Note: The relative correction factor is the final relative correction factor determined by the average value of the durability test results.

[0196] Using the peak area of ​​chebulic acid as a control, the contents of TeGG, chebulic acid, PGG, chebulic acid and ellagic acid were calculated by multiplying them by the correction factors of TeGG, chebulic acid, PGG, chebulic acid and ellagic acid respectively.

[0197] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for determining five components in Combretaceae medicinal materials based on a single-test, multiple-evaluation method, characterized in that, The method includes the following steps: 1) Prepare a mixed reference solution; the reference standards are chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloglucoside, 1,2,3,4,6-O-pentagalloglucoside and ellagic acid; 2) Preparation of the test solution: Take the medicinal material of the Combretaceae family, add solvent for extraction, then add solvent to make up the weight loss, shake well, filter, and obtain the test solution; the extraction method includes cold soaking, ultrasonic extraction or reflux extraction; the solvent is methanol with a volume percentage of 40-90%; 3) The mixed reference solution was diluted separately and then injected into the test solution into a high-performance liquid chromatograph for chromatographic detection. The chromatographic conditions were: C1... 18 The chromatographic column used methanol as mobile phase A and acid-water solution as mobile phase B, with the gradient elution program shown in the table below: ； The flow rate is 0.6-1.5 mL / min; the column temperature is 30-40℃; the detection wavelength is 190-400 nm; the injection volume is 3-10 μL; the acid aqueous solution is a 0.1%-0.3% acid aqueous solution; the acid aqueous solution is a phosphoric acid aqueous solution; 4) Based on the test results, obtain information about the Combretaceae medicinal material; the information includes component information and / or content information; The medicinal materials from the Combretaceae family mentioned are Terminalia chebula, Terminalia chebula var. hainanensis, and Terminalia chebula.

2. The method according to claim 1, characterized in that, The method further includes using chebulic acid as an internal reference to calculate the relative retention value and relative correction factor of the target compound; the target compound is chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloyl glucose, 1,2,3,4,6-O-pentagalloyl glucose and ellagic acid.

3. The method according to claim 1, characterized in that, The ultrasound power is 100-500W, the frequency is 20-100kHz, and the duration is 5-30min.

4. The method according to claim 1, characterized in that, The C 18 The chromatographic column was Phenomenex Luna C10. 18 Thermo Acclaim C 18 Or Welch Ultimate XB C 18 .

5. The method according to claim 1, characterized in that, The resolution R of each chromatographic peak is greater than 1.5, and the tailing factor T is 0.95-1.

05.

6. The method according to claim 2, characterized in that, When chebulic acid is used as an internal reference, the target compound is chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloyl glucose, 1,2,3,4,6-O-pentagalloyl glucose and ellagic acid.

7. The method according to claim 6, characterized in that, When chebulic acid was used as an internal reference, the target compound was located by chromatographic peaks using relative retention values; the relative retention value of chebulic acid was 0.52±5%; the relative retention value of 1,2,3,6-O-tetragalloylglucose was 0.47±5%; the relative retention value of 1,2,3,4,6-O-pentagalloylglucose was 0.90±5%; and the relative retention value of ellagic acid was 1.07±5%. When chebulic acid is used as an internal reference, the content of the target compound is calculated using relative correction factors; the relative correction factor for chebulic acid is 1.34±5%; the relative correction factor for 1,2,3,6-O-tetragalloylglucose is 0.81±5%; the relative correction factor for 1,2,3,4,6-O-pentagalloylglucose is 0.78±5%; and the relative correction factor for ellagic acid is 0.83±5%.

8. The application of the method according to any one of claims 1-7 in the detection of the contents of chebulic acid, chebulic acid, 1,2,3,6-O-tetragalloyl glucose, 1,2,3,4,6-O-pentagalloyl glucose and ellagic acid in Combretaceae medicinal materials.

9. The application of the method according to any one of claims 1-7 in evaluating the quality of Combretaceae medicinal materials, evaluating the authenticity of Combretaceae medicinal materials, or in the quality control of Combretaceae medicinal materials.

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