A method for determining contents of multiple components in Terminalia chebula and constructing characteristic fingerprint spectrum

Abstract

This invention relates to the field of traditional Chinese medicine analysis, and particularly to a method for determining the content of multiple components in Terminalia chebula and constructing its characteristic fingerprint spectrum. The method provided by this invention can simultaneously determine chebulin, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, punicalin (punicalin A&B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulin, urolithin M5, chebulin tannic acid, chebulin-lintannic acid, and ellagic acid in Terminalia chebula. It exhibits high sensitivity, good precision, repeatability, and stability, and can be used to detect the tannin content in the extract at each step of the tannin extraction process, as well as for related detection in the study and quality evaluation of tannin components in Terminalia chebula. It can also be used to construct the characteristic fingerprint spectrum of Terminalia chebula. This method can also be used for determining the content of tannin compounds and constructing their characteristic fingerprint spectrum in Terminalia chebula, which contains similar tannin compounds to Terminalia chebula.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine analysis, and particularly relates to a method for determining the contents of multiple components in Terminalia chebula and constructing a characteristic fingerprint spectrum. BACKGROUND

[0002] Terminalia chebula is the dried mature fruit of Terminalia chebula Retz. or Terminalia chebula Retz. var. tomentella Kurt. in the Combretaceae family, has the effects of astringing intestine to stop diarrhea, astringing lung to stop cough, and reducing fire to benefit the throat, and is mainly used for chronic diarrhea and dysentery, hematochezia, prolapse of the rectum, lung deficiency with cough, chronic cough, and sore throat and hoarseness.

[0003] Terminalia chebula contains a large amount of bioactive components, including polyphenols, flavonoids, polysaccharides, and terpenes, and the most abundant component is tannins, mainly including hydrolysable tannins such as gallic acid, corilagin, chebulagic acid, and ellagic acid. The analysis and determination of chemical substances in Terminalia chebula and the research on the characteristic fingerprint spectrum are helpful for the quality evaluation of medicinal materials, the discovery of active ingredients, and the research on the mechanism of action. However, the existing analysis and determination methods can only quantitatively determine one or a few tannin compounds in Terminalia chebula, and cannot comprehensively reflect the quality fluctuation of tannin components. SUMMARY

[0004] In view of the above technical problems, the present application provides a method for determining the contents of multiple components in Terminalia chebula and constructing a characteristic fingerprint spectrum and an application. The content determination method provided by the present application can accurately determine more than ten tannin compounds in Terminalia chebula at the same time, has high sensitivity, and has good precision, repeatability, and stability, and can be used not only for constructing a characteristic fingerprint spectrum of Terminalia chebula, but also for constructing a characteristic fingerprint spectrum of Ficus tikoua with common components.

[0005] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] A method for determining the contents of multiple components in Terminalia chebula, wherein Terminalia chebula is extracted by using a methanol aqueous solution, and the contents of the to-be-detected components are determined by using an ultra-performance liquid chromatography (UPLC), the to-be-detected components including chebulagic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin (corilagin A & B), chebulagic acid, 1,3,6-tri-O-galloyl-beta-D-glucose, chebuloside M5, chebulinic acid, chebulinic acid, and ellagic acid; and the chromatographic conditions of the ultra-performance liquid chromatography include:

[0007] a chromatographic column: a pentabromophenyl high-performance liquid chromatographic column;

[0008] The mobile phase A is 0.095%-0.105% v / v formic acid aqueous solution, and the mobile phase B is methanol, gradient elution, and the program of the gradient elution is as follows:

[0009]

[0010] The flow rate is 0.28-0.32 ml / min.

[0011] The detection wavelength is 250-290 nm.

[0012] The above chromatographic conditions can detect various tannin components in Terminalia chebula, including chebulagic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin (corilagin A & B), coralyne, 1,3,6-tri-O-galloyl-beta-D-glucose, chebulagic acid B, urolithin M5, chebulagic acid, chebulinic acid and ellagic acid, with high sensitivity and good precision, repeatability and stability, and can be used for quality evaluation of Terminalia chebula, content detection of tannin components in Terminalia chebula extract or extract, and construction of characteristic fingerprint spectrum of Terminalia chebula. The content determination method can also be used for content determination and characteristic fingerprint spectrum construction of tannin compounds in Terminalia chebula which contains similar tannin compounds.

[0013] In combination with the first aspect, the volume percentage concentration of the methanol aqueous solution is 40%-100%. Further preferably, the methanol is 100% v / v.

[0014] Preferably, the extraction method is ultrasonic extraction, the extraction temperature is 30-50 DEG C, and the extraction time is 10-30 min.

[0015] In combination with the first aspect, the five-bromophenyl high-performance liquid chromatography column is Cosmosil PBr Packed Column (2.1x100mm, 2.6μm). The chromatography column can not only separate large polar compounds under reverse phase conditions, but also be applied under 100% water conditions, suitable for polar compound separation; and has obvious advantages for cyclic compounds or amine compounds. For the test components in Terminalia chebula, the chromatography column has good separation effect.

[0016] In combination with the first aspect, the mobile phase A preferably uses 0.10% v / v formic acid aqueous solution.

[0017] In combination with the first aspect, the flow rate is 0.3 ml / min.

[0018] In combination with the first aspect, the chromatography conditions further include a column temperature of 30-60 DEG C; preferably 30 DEG C.

[0019] In combination with the first aspect, the mass-volume ratio of the Terminalia chebula to the methanol aqueous solution is 1:50-500 (g:ml). Preferably, 1:250-500 (g:ml) is adopted.

[0020] In combination with the first aspect, the content determination method comprises the following steps:

[0021] Step a, extracting the Terminalia chebula with a methanol aqueous solution to prepare a test sample solution;

[0022] Step b, preparing a control sample solution of the component to be determined;

[0023] Step c, determining the control sample solution and the test sample solution by the ultra-high performance liquid chromatography, and calculating the content of the component to be determined in the test sample by a standard curve method.

[0024] The second aspect of the present application further provides a characteristic fingerprint spectrum construction method of Terminalia chebula, comprising the following steps:

[0025] S1, preparing a control sample solution of the index component; the index component comprises chebulagic acid, gallic acid, 5-galloylshikimic acid, punicalagins (punicalagin A&B), corilagin, 1,3,6-tri-O-galloyl-beta-D-glucose, chebuloside, urolithin M5, Terminalia chebula tannin, Terminalia chebula gallate and ellagic acid;

[0026] S2, taking different batches of Terminalia chebula, respectively extracting with methanol to obtain test sample solutions of different batches of Terminalia chebula, determining the control sample solution and the test sample solution by the ultra-high performance liquid chromatography in the content determination method, recording the fingerprint spectrum, importing all the fingerprint spectra into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system software, taking the spectrum of one batch of Terminalia chebula as a reference spectrum, selecting a time width of 0.1 min, adopting a median method to perform peak matching with Mark peaks, performing mode recognition to obtain a fingerprint spectrum and a control fingerprint spectrum, selecting peaks with strong absorption signals, good separation degrees and good stabilities as characteristic peaks, and calculating the relative retention time and the relative peak area of each characteristic peak.

[0027] The characteristic fingerprint spectrum obtained by the characteristic fingerprint spectrum construction method of Terminalia chebula provided by the present application can obtain the spectrum information of the above-mentioned 12 kinds of tannin compounds, and can be used for rapid evaluation of the tannin compounds in Terminalia chebula, thereby providing data support for the quality research of Terminalia chebula.

[0028] Thirdly, the present invention also provides a characteristic fingerprint spectrum of Terminalia chebula, which contains 12 characteristic fingerprint peaks: in order, chebulic acid, gallic acid, 5-galloylshikimic acid, punicin A, punicin B, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulin, chebulic tannic acid, chebulin-lintannic acid, ellagic acid, and 4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyra nosyl)ellagic acid.

[0029] In conjunction with the third aspect, using the peak of Corilagin as the reference peak (H6), the relative retention times of each common peak and the reference peak are within ±5% of the specified values; the specified values ​​are: H1 peak (chebulic acid) 0.2220–0.2453, H2 peak (gallic acid) 0.4239–0.4685, H3 peak (5-gallicoylshikimic acid) 0.6435–0.7112, H4 peak (punicalin A) 0.7405–0.8185, H5 peak (punicalin B) 0.8456–0.9346, H6 peak (corilagin ... B) 0.6435–0.7112, H5 peak (5-gallicoylshikimic acid) 0.6435–0.7112, H5 peak (5-gallicoylshikimic acid) 0.6435–0.7112, H6 peak (5-gallicoylshikimic acid) 0.6435–0.7112, H4 peak (punicalin A) 0.7405–0.8185, H5 peak (punicalin B) 0.8456–0.9346, H6 peak (5-gallicoylshikimic acid) 0.6435–0.9112, H6 peak (5-gallicoylshikimic acid) 0.6435–0.9112, H6 peak (5-gallicoylshikimic acid) 0.643 The values ​​for H7 (1,3,6-tri-O-galloyl-β-D-glucose) and H8 (Terminol) are 1.167–1.290, H9 (Terminol) is 1.306–1.444, H10 (Terminol) is 1.501–1.659, H11 (Ellagic acid) is 1.532–1.693, and H12 (4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)el lagic acid) is 1.728–1.910.

[0030] Fourthly, the present invention also provides the application of the above-mentioned content determination method in the determination of tannin content and construction of characteristic fingerprint spectrum in Terminalia chebula.

[0031] The above-mentioned method for determining the content of Terminalia chebula provided by this invention can determine the content of tannins in Terminalia chebula, construct a characteristic fingerprint spectrum of Terminalia chebula, and quickly reflect the quality of Terminalia chebula.

[0032] The beneficial effects of this invention are as follows:

[0033] The method for determining the content of multiple components in Terminalia chebula established in this invention can simultaneously determine 12 tannin compounds in Terminalia chebula. Methodological validation has shown that this method has high sensitivity, good precision, repeatability, and stability, meeting the methodological requirements. This invention also establishes a method for constructing a characteristic fingerprint spectrum of Terminalia chebula and obtains 12 characteristic fingerprint peaks. Corilagin is selected as a reference peak, and the relative retention times of the common peaks in the characteristic fingerprint spectrum of Terminalia chebula are determined. The content determination method provided by this invention can be used for the determination of tannin compounds and the construction of characteristic fingerprint spectra in Terminalia chebula, which contains similar tannin compounds to Terminalia chebula, enabling quality evaluation and sample detection of both Terminalia chebula and Terminalia chebula. Attached Figure Description

[0034] Figure 1 Here are the liquid chromatograms of the different medicinal material test solutions in Example 3;

[0035] Figure 2 The TIC mass spectra of different medicinal material test solutions in Example 3 are shown below.

[0036] Figure 3 The fingerprint spectra of 35 batches of Terminalia chebula samples in Example 4 and the control fingerprint spectra are shown.

[0037] Figure 4 The fingerprint spectra of 28 batches of Terminalia chebula samples in Example 5 and the control fingerprint spectra are shown.

[0038] Figure 5 The above are liquid chromatograms of the reference solutions in Examples 1, 6, 7, and 8.

[0039] Figure 6 The images show liquid chromatograms obtained using different chromatographic columns in Comparative Example 1. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] Terminalia chebula contains a large number of bioactive components, the most abundant of which are tannins, mainly hydrolyzable tannins such as gallic acid, punicin, corilagin, and ellagic acid. Existing analytical methods can only quantitatively detect one or a few tannins in Terminalia chebula, which is insufficient to comprehensively reflect the quality fluctuations of its tannin components. To address this issue, this invention provides a method for determining the content of multiple components in Terminalia chebula and a method for constructing a characteristic fingerprint spectrum of Terminalia chebula, and also provides a method for constructing a characteristic fingerprint spectrum of Terminalia chebula based on this content determination method.

[0042] The present invention will be further described below with reference to several embodiments.

[0043] The main instruments used in the following embodiments:

[0044] Ultra-high performance liquid chromatograph (ACQUITY) H class plus (Waters Corporation, USA); Agilent Infinitiy II 1260 ultra-high performance liquid chromatograph tandem with 6550Q-TOF high resolution mass spectrometer (Agilent Technologies, USA); 0.001 g balance (ME204, METTLER, Switzerland); 0.001 g balance (New Classic MS, METTLER, Switzerland); intelligent ultrasonic cleaner (DL-720B, Shanghai Zhixin Instrument Co., Ltd.); high-speed refrigerated centrifuge (5430R, Eppendorf, Germany);

[0045] The reagents used in the following examples are:

[0046] Methanol (chromatographic grade, Sigma-Aldrich Trading Co., Ltd.); anhydrous ethanol (analytical grade, Tianjin Concord Technology Co., Ltd.); formic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); purified water (Guangzhou Watsons Food & Beverage Co., Ltd.); dimethyl sulfoxide (analytical grade, Tianjin Damao Chemical Reagent Factory).

[0047] The reagents used in the following examples:

[0048] Terminalia chebula (Beijing Tongrentang Tianjin Hexi Pharmacy Co., Ltd.); gallic acid (J05GB153704), punicin (J03HB186918), corilagin (A02GB143893), chebulic tannic acid (M29GB143373), chebulic tannic acid (J21HB174660), 1,3,6-tri-O-galloyl-β-D-glucose (J11GB154490), ellagic acid (S24D11G135548) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; chebulic acid, 4-galloshikimic acid, 5-galloshikimic acid, chebulic acid, and urolithin M5 were all prepared in our laboratory (purity greater than 98%).

[0049] The origin and source information of the test drugs used in the following examples, namely Terminalia chebula and Terminalia chebula, are shown in Table 1 (HZ represents Terminalia chebula, XQG represents Terminalia chebula).

[0050] Table 1. Origin and Source Information of Terminalia chebula and Terminalia chebula medicinal materials

[0051]

[0052]

[0053] The Terminalia chebula and Terminalia chebula powders used in the following examples are all powders obtained by pulverizing the above-mentioned medicinal materials and passing them through a No. 3 sieve.

[0054] Example 1

[0055] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0056] Step a, Preparation of the test solution

[0057] Accurately weigh 0.2g of Terminalia chebula powder and place it in a 50ml volumetric flask. Add an appropriate amount of methanol and extract by ultrasonication at 30℃ for 20min. After placing it at room temperature, dilute to the mark with methanol, shake well, and centrifuge an appropriate amount at 13700rpm for 10min. Take the supernatant as the test solution.

[0058] Step b: Preparation of the reference solution

[0059] Take appropriate amounts of 12 reference standards, accurately weigh them, dissolve the reference standards of chebulic acid and gallic acid in water, dissolve the reference standards of 4-galloshikimic acid, 5-galloshikimic acid, corilagin, and 1,3,6-tri-O-galloyl-β-D-glucose in methanol, and dissolve the reference standards of punicalin (punicalin A&B), chebulicin, urolithin M5, chebulic tannic acid, chebulicin tannic acid, and ellagic acid in dimethyl sulfoxide to prepare separate solutions. Reference stock solutions were prepared with concentrations of 10.014 mg / ml, 7.028 mg / ml, 1.022 mg / ml, 1.662 mg / ml, 1.092 mg / ml, 0.854 mg / ml, 4.048 mg / ml, 3.846 mg / ml, 1.190 mg / ml, 1.530 mg / ml, 1.864 mg / ml, and 0.5844 mg / ml.

[0060] Accurately measure appropriate amounts of stock solutions of chebulic acid, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, punicin (punicin A&B), chebulic acid, urolithin M5, chebulic tannic acid, chebulic lintannic acid, and ellagic acid, place them in a 10 ml volumetric flask, add dimethyl sulfoxide to the mark, and shake well to obtain a mixed reference solution. The mixed reference solution contained 500.7 μg / ml chebulic acid, 210.84 μg / ml gallic acid, 51.1 μg / ml 4-galloshikimic acid, 66.48 μg / ml 5-galloshikimic acid, 242.88 μg / ml punicalin (punicalin A&B), 65.52 μg / ml corilagin, 68.32 μg / ml 1,3,6-tri-O-galloyl-β-D-glucose, 230.76 μg / ml chebulin, 35.7 μg / ml urolithin M5, 183.6 μg / ml chebulic tannic acid, 186.4 μg / ml chebulin-tannic acid, and 175.32 μg / ml ellagic acid. A series of reference solutions with different concentrations were obtained through serial dilution.

[0061] Step c: The above mixed reference solution and test solution were analyzed using an Agilent Infinitiy II 1260 ultra-high performance liquid chromatograph connected in series with a 6550Q-TOF high resolution mass spectrometer. The chromatographic conditions were as follows:

[0062] Chromatographic column: Cosmosil PBr Packed Column (2.1×100mm, 2.6μm);

[0063] Column temperature: 30℃;

[0064] Injection volume: 2 μL;

[0065] Flow rate: 0.3 ml / min;

[0066] Mobile phase A is 0.10% v / v formic acid aqueous solution, and mobile phase B is methanol. Linear gradient elution is performed. The elution program is as follows:

[0067]

[0068] The detection wavelength is 270nm;

[0069] Mass spectrometry conditions were as follows: data was acquired in negative ion mode, with Auto MS / MS as the acquisition mode. Ion source parameters were set as follows: gas temperature 200℃; drying gas flow rate 12 L / min; nebulization pressure 40 psi; sheath gas temperature 350℃; nozzle voltage -1.5 kV; capillary voltage -4.0 kV; fragmentor voltage 390 V; and the mass-to-charge ratio (m / z) range scanned by the TOF analyzer was [MS / MS]. 1 For 100–1500, MS 2 The value is 50–1500. Automatic selection in MS via collision-induced dissociation. 1 The top three ions in intensity in the spectrum triggered MS / MS fragmentation at a collision energy of 30 V. Data acquisition was performed using Agilent MassHunter Workstation DataAcquisition software.

[0070] Test Example 1

[0071] The present invention provides a methodological investigation of the multi-component content determination method in Example 1, wherein:

[0072] (1) Study on linear relationship and limit of detection and limit of quantitation

[0073] A series of mixed reference solutions were prepared according to step b in Example 1. Peak areas were determined using ultra-high performance liquid chromatography (UHPLC) under the same chromatographic conditions as step c in Example 1. Two parallel injections were performed. A regression equation was plotted with the concentration of each compound reference standard as the abscissa (x, μg / ml) and the corresponding peak area as the ordinate. The limits of detection (LOD) and quantitation (LOQ) were set for chebulic acid, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, punicalin (punicalin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic tannic acid, chebuliclin tannic acid, and ellagic acid. The results are shown in Table 2.

[0074]

[0075] As shown in Table 2, each component exhibits good linearity within its respective concentration range (r0). 2 The detection limit (≥0.999) has a concentration range of 0.0998–0.9779 μg / ml, and the quantitation limit (LOQ) has a concentration range of 0.2559–1.956 μg / ml, indicating that the method for determining the content of multiple components in Terminalia chebula provided by this invention has high sensitivity.

[0076] (2) Precision test

[0077] (a) Intraday precision test

[0078] Accurately pipette the same sample solution and inject it six times. Measure the peak area using ultra-high performance liquid chromatography (UHPLC) under the same chromatographic conditions as step c in Example 1. Calculate the RSD value based on the peak area, and the results are shown in Table 3.

[0079] Table 3. Precision test results within 3 days (n=6)

[0080]

[0081] As shown in Table 3, the intra-day precision RSD of the method for determining the content of multiple components in Terminalia chebula provided in Example 1 is <1.0%.

[0082] (b) Daytime precision test

[0083] For three consecutive days, the same sample solution was precisely pipetted each day and injected six times. The peak area was determined by ultra-high performance liquid chromatography (UHPLC) under the same chromatographic conditions as step c in Example 1. The results are shown in Table 4.

[0084] Table 4. Results of daytime precision tests (n=3)

[0085]

[0086] As shown in Table 4, the inter-day precision (RSD) of the method for determining the content of multiple components in Terminalia chebula provided in Example 1 is <2.7%, indicating good precision.

[0087] (3) Repeatability test

[0088] Six parallel test solutions of Terminalia chebula powder were prepared according to step a in Example 1. The contents of each compound in these test solutions were determined by ultra-high performance liquid chromatography (UHPLC), under the same chromatographic conditions as step c in Example 1. The contents of chebulin, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, punicalin (punicalin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulin, urolithin M5, chebulin tannic acid, chebulin-lintannic acid, and ellagic acid were calculated using a standard curve, and the RSD values ​​were also calculated. The results are shown in Table 5.

[0089] As shown in Table 5, the RSDs of the following compounds in this experiment were all less than 3.0%, indicating that the detection method had good repeatability: 41.48 mg / g chebulic acid, 12.65 mg / g gallic acid, 2.530 mg / g 4-galloshikimic acid, 3.760 mg / g 5-galloshikimic acid, 9.692 mg / g punicin (punicin A&B), 3.809 mg / g corilagin, 3.467 mg / g 1,3,6-tri-O-galloyl-β-D-glucose, 17.56 mg / g chebulicin, 2.707 mg / g urolithin M5, 9.985 mg / g chebulic tannic acid, 7.878 mg / g chebulic tannic acid, and 28.76 mg / g ellagic acid.

[0090] (4) Stability test

[0091] The test solution was stored at 10°C. A precise sample of the same solution was taken at 0, 2, 4, 6, 8, 10, and 12 hours and analyzed by ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions were the same as in step c of Example 1. The RSD values ​​were calculated based on the peak areas of chebulic acid, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, punicalin (punicalin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic tannic acid, chebuliclin tannic acid, and ellagic acid. The results are shown in Table 6.

[0092] As shown in Table 6, the stability test RSD of the liquid chromatography conditions for the method for determining the content of multiple components in Terminalia chebula provided in Example 1 is <1.2%, indicating that the test solution has good stability within 12 hours at room temperature.

[0093]

[0094]

[0095] (5) Recovery test

[0096] Accurately weigh approximately 0.1 g of Terminalia chebula powder and add it to 16.6 ml of a mixed reference solution with a mass equivalent to that of each component in the sample (Terminidin 300.4 μg / ml, Gallic acid 84.34 μg / ml, 4-Galloylshikimic acid 18.40 μg / ml, 5-Galloylshikimic acid 29.92 μg / ml, Punicin (Punicin A & B) 72.86 μg / ml, Corilagin 32.76 μg / ml, 1,3,6-Tri-O-Galloyl-β-D-glucose 20.50 μg / ml, Terminalia chebulin 115.4 μg / ml, Urolithin M5 17.85 μg / ml, Terminalia chebulin tannic acid 64.26 μg / ml, Terminalia chebulin tannic acid 55.92 μg / ml, Ellagic acid 175.3 μg / ml).

[0097] Six chebula sample solutions were prepared, and each sample was measured twice in parallel. The solutions were analyzed using ultra-high performance liquid chromatography (UHPLC) under the same chromatographic conditions as step c in Example 1. The accuracy of the method was assessed by calculating the recovery rate and RSD values ​​using the recovery formula.

[0098] The formula for recovery rate is as follows:

[0099]

[0100] The results are shown in Table 7.

[0101] As shown in Table 7, the average recovery rate of the liquid chromatography method for determining the content of multiple components in Terminalia chebula provided in Example 1 was 97.41-113.4%, and the RSD values ​​were all less than 6.5%, indicating that the recovery rate of each compound basically met the requirements, and the method is accurate and feasible.

[0102]

[0103]

[0104]

[0105] As shown in Tables 2-7, the method for determining the content of multiple components in Terminalia chebula provided in Example 1 basically meets the requirements of methodological research and can be used for the quantitative analysis of Terminalia chebula.

[0106] Example 2

[0107] In this embodiment, the content of tannins in different batches of Terminalia chebula was determined using the content determination method described in Example 1. The steps were the same as in Example 1, and the results are shown in Table 8.

[0108] Example 3

[0109] In this embodiment, the tannins in Terminalia chebula were detected using the content determination method described in Example 1.

[0110] Step a, Preparation of the test solution: Same as in Example 1, except that Terminalia chebula powder is replaced with Terminalia chebula powder;

[0111] Step b, Preparation of the reference solution: Same as in Example 1;

[0112] Step c is the same as in Example 1, and the results are shown in Table 9.

[0113] The liquid chromatograms of the test solutions of each medicinal material are as follows: Figure 1 As shown, the TIC mass spectrum is as follows: Figure 2 As shown in Table 10, the qualitative test results are as follows.

[0114]

[0115]

[0116]

[0117] Example 4

[0118] This invention provides a method for constructing a characteristic fingerprint spectrum of Terminalia chebula, comprising the following steps:

[0119] Step 1: Prepare the test solution and mixed reference solution of Terminalia chebula powder according to the preparation methods of the test solution and reference solution in Example 1;

[0120] Step 2: Same as step c in Example 1, record the feature fingerprint spectrum.

[0121] The data were imported into the 2012 version of the Chinese herbal medicine chromatographic fingerprint similarity evaluation system for processing. Using the chromatogram of Terminalia chebula HZ35 as a reference chromatogram, 12 main characteristic peaks with strong absorption signals and good resolution and stability were selected as common peaks. (See...) Figure 3The similarity between the fingerprint chromatograms of Terminalia chebula and the control fingerprint chromatogram was calculated. The results showed that the similarity between the 35 batches of Terminalia chebula samples and the control chromatogram was greater than 0.66, indicating a large difference between batches of Terminalia chebula. Based on the determination of the relative retention times of each compound in the fingerprint chromatograms of the 35 batches of Terminalia chebula, 12 common chromatographic peaks with larger peak areas, better peak shapes, and better resolution were selected as characteristic fingerprint peaks. A standard control fingerprint chromatogram was generated, with peak H6 as the reference peak. The relative retention times and relative peak areas of the 12 common chromatographic peaks are shown in Tables 11 and 12. By comparing the chromatograms with those of the reference standards, the characteristic fingerprint peaks were identified as chebulic acid (H1), gallic acid (H2), 5-galloylshikimic acid (H3), punicalin A (H4), punicalin B (H5), cristatin (H6), 1,3,6-tri-O-galloyl-β-D-glucose (H7), chebulic acid (H8), chebulic tannic acid (H9), chebulic lintannic acid (H10), ellagic acid (H11), and 4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (H12).

[0122]

[0123]

[0124]

[0125] Example 5

[0126] This invention provides a method for constructing a characteristic fingerprint spectrum of Terminalia chebula, comprising the following steps:

[0127] Step 1: Prepare the test solution and mixed reference solution of Terminalia chebula powder according to the preparation methods of the test solution and reference solution in Example 1.

[0128] Step 2: Same as step c in Example 1, record the feature fingerprint spectrum.

[0129] The data were imported into the 2012 version of the Chinese herbal medicine chromatographic fingerprint similarity evaluation system for processing. Using the chromatogram of Terminalia chebula XQG24 as the reference chromatogram, 11 main characteristic peaks with strong absorption signals and good resolution and stability were selected as common peaks. (See...) Figure 4The similarity between the UPLC chromatograms of each sample and the control fingerprint chromatogram was calculated. The results showed that the similarity between the 28 batches of Terminalia chebula samples and the control chromatogram was greater than 0.86, indicating good similarity. Based on the determination of the relative retention times of each compound in the fingerprint chromatograms of the 28 batches of Terminalia chebula medicinal materials, 11 common chromatographic peaks with larger peak areas, better peak shapes, and better resolution were selected as characteristic fingerprint peaks. A standard control fingerprint chromatogram was generated, with peak X5 (Corilagin) as the reference peak. The relative retention times and relative peak areas of the 11 common chromatographic peaks are shown in Tables 13 and 14. By comparing the chromatograms with those of the reference standards, the characteristic fingerprint peaks were identified as chebulic acid (X1), gallic acid (X2), punicalin A (X3), punicalin B (X4), corilagin (X5), 1,3,6-tri-O-galloyl-β-D-glucose (X6), chebulic acid (X7), urolithin M5 (X8), chebulic tannic acid (X9), chebulic tannic acid (X10), and ellagic acid (X11).

[0130] Using the peak of Corilagin as a reference peak (X5), the relative retention times of each common peak and the reference peak were within ±5% of the specified values; the specified values ​​were: X1 peak (chebulic acid) 0.2232-0.2466, X2 peak (gallic acid) 0.4239-0.4686, X3 peak (punicalin A) 0.7401-0.818, X4 peak (punicalin B) 0.8442-0.933, and X5 peak (corilagin) 1. .000, X6 peak (1,3,6-tri-O-galloyl-β-D-glucose) is 1.057-1.168, X7 peak (Terminol) is 1.167-1.290, X8 peak (urolithin M5) is 1.201-1.327, X9 peak (Terminol tannic acid) is 1.307-1.444, H10 peak (Terminol tannic acid) is 1.503-1.662, and X11 peak (ellagic acid) is 1.533-1.694.

[0131]

[0132]

[0133]

[0134] Example 6

[0135] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0136] Step a, Preparation of the test solution: Same as in Example 1;

[0137] Step b, Preparation of the reference solution: Same as in Example 1;

[0138] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph. The chromatographic conditions were basically the same as in Example 1, except that the column temperature was 40°C.

[0139] Example 7

[0140] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0141] Step a, Preparation of the test solution: Same as in Example 1;

[0142] Step b, Preparation of the reference solution: Same as in Example 1;

[0143] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph. The chromatographic conditions were basically the same as in Example 1, except that the column temperature was 50°C.

[0144] Example 8

[0145] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0146] Step a, Preparation of the test solution: Same as in Example 1;

[0147] Step b, Preparation of the reference solution: Same as in Example 1;

[0148] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph. The chromatographic conditions were basically the same as in Example 1, except that the column temperature was 60°C.

[0149] The liquid chromatograms of the test solutions in Examples 1, 6, 7, and 8 are as follows: Figure 5 As shown, the peaks of 13 tannin compounds can be separated under column temperatures ranging from 30 to 60°C, with better peak separation at 30°C.

[0150] Example 9

[0151] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0152] Step a, Preparation of test solution: Accurately weigh 0.2g of Terminalia chebula powder and place it in a 25ml volumetric flask. Add an appropriate amount of methanol and extract by ultrasonication at 30℃ for 20min. After standing at room temperature, dilute to the mark with methanol, shake well, and centrifuge an appropriate amount at 13700rpm for 10min. Take the supernatant as the test solution. Prepare test solutions with 60% v / v, 80% v / v and 100% v / v as extraction solvents in the same way.

[0153] Step b, preparation of the reference solution: same as in Example 1.

[0154] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, under the same chromatographic conditions as in Example 1.

[0155] The peak areas of each component in the liquid chromatograms of each test solution are shown in Table 15.

[0156] Table 15 Peak areas of tannin components in the test solution obtained under different extraction solvents.

[0157]

[0158] As shown in Table 15, when methanol is used as the extraction solvent, the peak area of ​​tannins is the highest, and the extraction effect is the best.

[0159] Example 10

[0160] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0161] Step a, Preparation of test solution: Accurately weigh 1, 0.2, 0.2, and 0.1 g of Terminalia chebula powder and place them in volumetric flasks of 50 ml, 25 ml, 50 ml, and 50 ml respectively. Accurately add an appropriate amount of methanol to each flask, extract by ultrasonication at 30℃ for 20 min, and after standing at room temperature, dilute to the mark with methanol and shake well to obtain solutions with material-to-liquid ratios of 1:50, 1:125, 1:250, and 1:500. Take an appropriate amount of each solution and centrifuge at 13700 rpm for 10 min. Take the supernatant as the test solution.

[0162] Step b, preparation of the reference solution: same as in Example 1.

[0163] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, under the same chromatographic conditions as in Example 1.

[0164] The peak areas of each component in the liquid chromatograms of each test solution are shown in Table 16.

[0165] Table 16 Peak areas of tannin components in the test solution obtained at different solid-liquid ratios

[0166]

[0167] As shown in Table 16, the peak areas of tannin components were higher and the extraction effect was better when the material-to-liquid ratio was 1:250 and 1:500. The preferred material-to-liquid ratio was 1:250.

[0168] Example 11

[0169] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0170] Step a, Preparation of test solution: Accurately weigh 0.2g of Terminalia chebula powder and place it in a 50ml volumetric flask. Add an appropriate amount of methanol and extract by ultrasonication at 30℃ for 10, 20 and 30 min respectively. After being placed at room temperature, dilute to the mark with methanol and shake well. Take an appropriate amount of each sample and centrifuge at 13700rpm for 10 min. Take the supernatant as the test solution.

[0171] Step b, preparation of the reference solution: same as in Example 1.

[0172] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, under the same chromatographic conditions as in Example 1.

[0173] The peak areas of each component in the liquid chromatograms of each test solution are shown in Table 17.

[0174] Table 17 Peak areas of tannin components in the test sample solution obtained at different ultrasonic times.

[0175]

[0176] As shown in Table 17, the peak areas of tannin components were higher when the ultrasonic time was 20 min and 30 min, indicating better extraction effect. Therefore, the ultrasonic time of 20 min was selected.

[0177] Example 12

[0178] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0179] Step a, Preparation of test solution: Accurately weigh 0.2g of Terminalia chebula powder and place it in a 50ml volumetric flask. Add an appropriate amount of methanol and extract by ultrasonication at 30, 40 and 50℃ for 20min respectively. After being placed at room temperature, dilute to the mark with methanol and shake well. Take an appropriate amount of each sample and centrifuge at 13700rpm for 10min. Take the supernatant as the test solution.

[0180] Step b, preparation of the reference solution: same as in Example 1.

[0181] Step c: The above mixed reference solution and test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, under the same chromatographic conditions as in Example 1.

[0182] The peak areas of each component in the liquid chromatograms of each test solution are shown in Table 18.

[0183] Table 18 Peak areas of tannin components in the test solution obtained at different ultrasonic temperatures.

[0184]

[0185] As shown in Table 18, the tannins had the highest peak area and the best extraction effect when the ultrasonic temperature was 30℃.

[0186] Comparative Example 13

[0187] This invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

[0188] Step a, Preparation of the test solution: Same as the preparation of the reference solution in Example 1;

[0189] Step b: The above-mentioned test solution was analyzed using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph under the following chromatographic conditions:

[0190] Chromatographic columns: ZORBAX SB-C18 (4.6 x 100 mm, 1.8 μm), ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm) or Cosmosil PBr Packed Column (2.1 x 100 mm, 2.6 μm);

[0191] Column temperature: 30℃

[0192] Injection volume: 2 μL;

[0193] Flow rate: 0.3 ml / min;

[0194] Mobile phase A is 0.1% v / v formic acid aqueous solution, and mobile phase B is methanol. Linear gradient elution is performed. The elution program is as follows:

[0195]

[0196] The chromatogram obtained when the column is ZORBAX SB-C18 is as follows: Figure 6The chromatogram is shown in Figure A; the chromatogram of the column is ACQUITYUPLC HSS T3 as shown in Figure A. Figure 6 The chromatogram is shown in Figure B; the chromatogram for a Cosmosil PBr Packed Column is shown in Figure B. Figure 6 As shown in Figure C.

[0197] Depend on Figure 6 It is evident that the Cosmosil PBr Packed Column is more suitable for the separation of tannin compounds (with similar polarity) in Terminalia chebula and achieves better separation.

[0198] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the content of multiple components in Terminalia chebula, characterized in that, Terminalia chebula was extracted with methanol, and the content of the analytes was determined by ultra-high performance liquid chromatography (UHPLC). The analytes included chebulic acid, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, punicin A, punicin B, corilagin, and 1,3,6-tri- O -Galloyl- β -D-glucose, chebulin, urolithin M5, chebulic tannic acid, chebulin-lin tannic acid, and ellagic acid; the extraction method is ultrasonic extraction; the chromatographic conditions of the ultra-high performance liquid chromatography include: Chromatographic column: Pentabromophenyl high performance liquid chromatography column; Mobile phase: Mobile phase A is a 0.095%–0.105% v / v formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is performed, and the gradient elution procedure is as follows: The flow rate was 0.28~0.32 ml / min; The detection wavelength is 250~290nm.

2. The content determination method according to claim 1, characterized in that, The extraction temperature is 30~50 ℃, and the extraction time is 10~30 min.

3. The content determination method according to claim 1, characterized in that, The pentabromophenyl high-performance liquid chromatography column is a Cosmosil PBr Packed Column with dimensions of 2.1 × 100 mm and 2.6 μm.

4. The content determination method according to claim 1, characterized in that, Mobile phase A is a 0.10% v / v aqueous formic acid solution; and / or The flow rate was 0.3 ml / min.

5. The content determination method according to claim 1, characterized in that, The chromatographic conditions also include a column temperature of 30-60 °C.

6. The content determination method according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step a: Extract Terminalia chebula with methanol-water solution to prepare test solution; Step b: Prepare a reference solution of the analyte; Step c: Use the ultra-high performance liquid chromatography method to determine the content of the reference solution and the test solution, and use the standard curve method to calculate the content of the analyte in the test sample.

7. A method for constructing a characteristic fingerprint spectrum of Terminalia chebula, characterized in that, Includes the following steps: S1. Prepare reference solutions for the indicator components; the indicator components include chebulic acid, gallic acid, 4-galloshikimic acid, 5-galloshikimic acid, punicetoxin A, punicetoxin B, corilagin, 1,3,6-tri- O -Galloyl- β -D-glucose, chebulin, urolithin M5, chebulic tannic acid, chebulic lintannic acid and ellagic acid; S2. Take different batches of Terminalia chebula and extract them with methanol to obtain test solutions of different batches of Terminalia chebula. Use the ultra-high performance liquid chromatography method in the content determination method of claim 1 to determine the content of the reference solution and the test solution, record the characteristic fingerprint spectrum, and import all the characteristic fingerprint spectrum into the software of the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. Using the spectrum of a batch of Terminalia chebula as the reference spectrum, select the peak with strong absorption signal and good separation and stability as the characteristic peak, and calculate the relative retention time and relative peak area of ​​each characteristic peak.

8. The method for constructing a feature fingerprint map according to claim 7, characterized in that, The characteristic fingerprint spectrum contains 12 characteristic fingerprint peaks: in order, chebulic acid, gallic acid, 5-galloshikimic acid, punicin A, punicin B, corilagin, 1,3,6-tri- O -Galloyl- β -D-glucose, chebulin, urolithin M5, chebulic tannic acid, chebulic tannic acid and ellagic acid.

9. The method for constructing a feature fingerprint map according to claim 8, characterized in that, The characteristic fingerprint spectrum uses the Corilagin peak as a reference peak, and the relative retention time of each common peak and the reference peak is within ±5% of a specified value; the specified values ​​are: H1 peak 0.2220–0.2453, H2 peak 0.4239–0.4685, H3 peak 0.6435–0.7112, H4 peak 0.7405–0.8185, H5 peak 0.8456–0.9346, H6 peak 1.000, H7 peak 1.056–1.167, H8 peak 1.167–1.290, H9 peak 1.306–1.444, H10 peak 1.501–1.659, H11 peak 1.532–1.693, and H12 peak 1.728–1.

910.

10. The application of the content determination method according to any one of claims 1 to 6 in the content determination and characteristic fingerprint construction of tannin components in Terminalia chebula.

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