CN116642988A - Method for measuring content of multiple components in myrobalan and constructing characteristic fingerprint - Google Patents

Abstract

The invention relates to the field of traditional Chinese medicine analysis, in particular to a method for measuring the content of multiple components in myrobalan and constructing a characteristic fingerprint. The content determination method provided by the invention can be used for simultaneously determining the myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid in the myrobalan, has high sensitivity and good precision, repeatability and stability, can be used for detecting the content of the tannins in the extracting solution of each step in the tannins component extraction process, and can be used for carrying out related detection in the research and quality evaluation of the tannins components of the myrobalan, and can be used for constructing the characteristic fingerprint of the myrobalan. The method can also be used for measuring the content of tannins in fructus Chebulae containing similar tannins and constructing characteristic fingerprint.

Description

A method for determining the content of multiple components in Terminalia chebula and constructing characteristic fingerprints

Technical Field

The invention relates to the field of traditional Chinese medicine analysis, and in particular to a method for determining the contents of multiple components in terminalia chebula and constructing a characteristic fingerprint spectrum.

Background Art

The Chinese medicine Terminalia chebula Retz is the dried mature fruit of Terminalia chebula Retz. or Terminalia chebula Retz.var.tomentella Kurt. of the Combretaceae family. It has the effects of astringing the intestines and stopping diarrhea, astringing the lungs and stopping coughs, reducing fire and relieving sore throats. It is mainly used for long-term diarrhea and dysentery, rectal prolapse due to bloody stools, lung deficiency, wheezing and coughing, long-term coughs, and sore throats and hoarseness.

Terminalia chebula contains a large number of bioactive ingredients, including polyphenols, flavonoids, polysaccharides, terpenes, etc. Among them, the most abundant are tannin compounds, mainly hydrolyzable tannins, such as gallic acid, punicalagin, corilagin, ellagic acid, etc. The analysis and content determination of chemical substances in Terminalia chebula and the study of characteristic fingerprints are helpful for the quality evaluation of medicinal materials, the discovery of active ingredients, and the study of mechanism of action. However, the existing analytical detection methods can only quantitatively detect one or a few tannin compounds in Terminalia chebula, which is not enough to more comprehensively reflect the quality fluctuation of its tannin components.

Summary of the invention

In view of the above technical problems, the present invention provides a method and application for determining the content of multiple components in Terminalia chebula and constructing a characteristic fingerprint. The content determination method provided by the present invention can accurately detect more than ten tannin compounds in Terminalia chebula at the same time, with high sensitivity and good precision, repeatability and stability. It can be used not only to construct a characteristic fingerprint of Terminalia chebula, but also to construct a characteristic fingerprint of Cibotium barometz which has common components with Terminalia chebula.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

A method for determining the contents of multiple components in terminalia chebula comprises extracting terminalia chebula with a methanol aqueous solution, and determining the contents of the components to be determined by ultra-high performance liquid chromatography (UPLC), wherein the components to be determined include terminalia chebula acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, terminalia chebula, urolithin M5, terminalia tannic acid, terminalia chebula tannic acid and ellagic acid; and the chromatographic conditions of the ultra-high performance liquid chromatography include:

Chromatographic column: Pentabromophenyl HPLC column;

Mobile phase: Mobile phase A is 0.095% to 0.105% v/v formic acid aqueous solution, mobile phase B is methanol, gradient elution, the gradient elution procedure is as follows:

Flow rate: 0.28～0.32ml/min;

Detection wavelength: 250～290nm;

The above chromatographic conditions can detect a variety of tannin components in myrobalan, including myrobalan acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, myrobalanine, urolithin M5, myrobalan tannic acid, myrobalan tannic acid and ellagic acid, with high sensitivity and good precision, repeatability and stability, and can be used for the quality evaluation of myrobalan, the content detection of tannin components in myrobalan extracts or extracts, and the construction of characteristic fingerprints of myrobalan. The content determination method can also be used for the content determination of tannin compounds in the fruit of the citrus aurantium containing similar tannin compounds as myrobalan and the construction of characteristic fingerprints.

In combination with the first aspect, the volume percentage concentration of the methanol aqueous solution is 40% to 100%, and more preferably methanol, that is, 100% v/v methanol.

Preferably, the extraction method is ultrasonic extraction, the extraction temperature is 30 to 50° C., and the extraction time is 10 to 30 minutes.

In combination with the first aspect, the pentabromophenyl high performance liquid chromatography column is a Cosmosil PBr Packed Column (2.1×100 mm, 2.6 μm). The chromatographic column can not only separate highly polar compounds under reverse phase conditions, but also can be used under 100% water conditions, and is suitable for the separation of polar compounds; and has obvious advantages for cyclic compounds or amine compounds. For the components to be tested in Terminalia chebula, the chromatographic column has a good separation effect.

In combination with the first aspect, the mobile phase A is preferably a 0.10% v/v formic acid aqueous solution.

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

In combination with the first aspect, the chromatographic conditions also include a column temperature of 30-60°C, preferably 30°C.

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).

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

Step a, extracting Terminalia chebula with methanol-water solution to prepare a test solution;

Step b, preparing a reference solution of the component to be tested;

Step c, using the ultra-high performance liquid chromatography method to measure the reference solution and the test solution, and using the standard curve method to calculate the content of the index component to be measured in the test solution.

In a second aspect, the present invention also provides a method for constructing a characteristic fingerprint of Terminalia chebula, comprising the following steps:

S1. Prepare a reference solution of the index components; the index components include chebulic acid, gallic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic acid, chebulic acid and ellagic acid;

S2. Take different batches of Terminalia chebula and extract them with methanol respectively to obtain test solutions of different batches of Terminalia chebula, measure the reference solution and the test solution with the ultra high performance liquid chromatography in the above-mentioned content determination method, record the fingerprint, import all the fingerprints into the Chinese medicine chromatographic fingerprint similarity evaluation system software, take the spectrum of a batch of Terminalia chebula as the reference spectrum, select the time width of 0.1min, use the median method to perform peak matching with Mark peak, perform pattern recognition on it to obtain fingerprint spectrum and reference fingerprint spectrum, select peaks with strong absorption signal, good separation and stability as characteristic peaks, and calculate the relative retention time and relative peak area of each characteristic peak.

The characteristic fingerprint constructed by the characteristic fingerprint construction method of Terminalia chebula provided by the present invention can obtain the spectrum information of the above-mentioned 12 tannin compounds, which can be used for rapid evaluation of the tannin compounds in Terminalia chebula and provide data support for the quality research of Terminalia chebula.

In the third aspect, the present invention also provides a terminalia chebula characteristic fingerprint, which comprises 12 characteristic fingerprint peaks: terminalia chebula acid, gallic acid, 5-galloylshikimic acid, punicalagin A, punicalagin B, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, terminalia chebula, terminalia tannic acid, terminalia chebula tannic acid, ellagic acid and 4-O-(3",4"-Di-O-galloyl-α-L-rhamnopyra nosyl)ellagic acid.

In combination with the third aspect, the peak of corilagin is used as the reference peak (H6), and the relative retention time of each common peak and the reference peak is within the range of ±5% of the specified value; the specified value is: H1 peak (chebulic acid) is 0.2220–0.2453, H2 peak (gallic acid) is 0.4239–0.4685, H3 peak (5-gallic acid ylshikimic acid) is 0.6435–0.7112, H4 peak (punicalagin A) is 0.7405–0.8185, H5 peak (punicalagin B) is 0.8456–0.9346, H6 peak (corilagin The peak values of H1 (H2) and H3 (H4) were 1.000, H7 (1,3,6-tri-O-galloyl-β-D-glucose) were 1.056–1.167, H8 (chebulic acid) were 1.167–1.290, H9 (chebulic acid) were 1.306–1.444, H10 (chebulic acid) were 1.501–1.659, H11 (ellagic acid) were 1.532–1.693, and H12 (4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)el lagic acid) were 1.728–1.910.

In a fourth aspect, the present invention also provides the application of the above-mentioned content determination method in the determination of tannin components in western green fruit and the construction of characteristic fingerprints.

The method for determining the content of terminalia chebula provided by the present invention can be used to determine the content of tannin components in terminalia chebula, construct a characteristic fingerprint of terminalia chebula, and quickly reflect the quality of terminalia chebula.

The beneficial effects of the present invention are:

The method for determining the content of multiple components in terminalia chebula established by the present invention can simultaneously determine 12 tannin compounds in terminalia chebula. After methodological verification, the method for determining the content is highly sensitive and has good precision, repeatability and stability, and meets the methodological requirements. The present invention also establishes a method for constructing a characteristic fingerprint of terminalia chebula, and obtains 12 characteristic fingerprint peaks of terminalia chebula. The present invention selects corilagin as a reference peak, and determines the relative retention time of the common peaks of the terminalia chebula characteristic fingerprint. The method for determining the content of tannin compounds in citrus aurantium containing similar tannin compounds to terminalia chebula and the construction of characteristic fingerprints can realize the quality evaluation of terminalia chebula and citrus aurantium and sample detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a liquid chromatogram of different medicinal material test sample solutions in Example 3;

FIG2 is a TIC mass spectrogram of different medicinal material test solutions in Example 3;

FIG3 is a characteristic fingerprint of 35 batches of Terminalia chebula samples and a control fingerprint in Example 4;

Figure 4 is the characteristic fingerprints and control fingerprints of 28 batches of Xiqing fruit samples in Example 5;

Fig. 5 is a liquid chromatogram of the reference substance solution in Examples 1, 6, 7, and 8;

FIG6 is a liquid chromatogram obtained using different chromatographic columns in Comparative Example 1.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

Terminalia chebula contains a large number of biologically active ingredients, among which the most abundant are tannin compounds, mainly hydrolyzable tannins, such as gallic acid, punicalagin, corilagin, ellagic acid, etc. Existing analytical detection methods can only quantitatively detect one or a few tannin compounds in Terminalia chebula, which is not enough to more comprehensively reflect the quality fluctuation of its tannin components. In view of this problem, the embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula and a method for constructing a characteristic fingerprint of Terminalia chebula, and provides a method for constructing a characteristic fingerprint of Cibotium barometz based on the content determination method.

The present invention is further described below with reference to a number of embodiments.

The main instruments used in the following examples are:

Ultra-high performance liquid chromatography (ACQUITY H class plus, Waters, USA); Agilent Infinitiy II 1260 ultra-high performance liquid chromatograph connected in series with 6550Q-TOF high-resolution mass spectrometer (Agilent, USA); 1/10,000 balance (ME204, METTLER, Switzerland); 1/100,000 balance (New Classic MS, METTLER, Switzerland); intelligent ultrasonic cleaner (DL-720B, Shanghai Zhixin Instrument Co., Ltd.); high-speed refrigerated centrifuge (5430R, Eppendorf, Germany);

The reagents used in the following examples are:

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 and Beverage Co., Ltd.); dimethyl sulfoxide (analytical grade, Tianjin Damao Chemical Reagent Factory).

The reagents used in the following examples are:

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

The test drugs used in the following examples: the origin and source information of the medicinal materials of Terminalia chebula and XQG are shown in Table 1 (HZ represents Terminalia chebula, and XQG represents XQG).

Table 1 Origin and source information of Terminalia chebula and Cibotium barometz

The chebula and citronella powders used in the following examples are all powders obtained by pulverizing the above medicinal materials and passing through a No. 3 sieve.

Example 1

The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution

Take 0.2g of Terminalia chebula powder, weigh accurately, place in a 50ml volumetric flask, add appropriate amount of methanol, ultrasonically extract at 30℃ for 20min, leave at room temperature, dilute to scale with methanol, shake well, take appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution;

Step b, preparation of reference solution

Take appropriate amount of 12 kinds of reference substances, weigh accurately, dissolve the reference substances of chebulic acid and gallic acid in water, dissolve the reference substances of 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose in methanol, dissolve the reference substances of punicalagin (punicalagin A & B), chebulic acid, urolithin M5, chebulic acid, chebulic acid, ellagic acid in dimethyl sulfoxide, and prepare respectively. The reference substance stock solutions are 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.

Accurately measure an appropriate amount of reference substance stock solutions of chebulic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, punicalagin (punicalagin A & B), chebulic acid, urolithin M5, chebulic acid, chebulic acid tannic acid, and ellagic acid, place in a 10 ml volumetric flask, add dimethyl sulfoxide to make up to the mark, shake well, and obtain a mixed reference substance solution. The mixed reference solution contained 500.7μg/ml of terminalic acid, 210.84μg/ml of gallic acid, 51.1μg/ml of 4-galloylshikimic acid, 66.48μg/ml of 5-galloylshikimic acid, 242.88μg/ml of punicalagin (punicalagin A&B), 65.52μg/ml of corilagin, 68.32μg/ml of 1,3,6-tri-O-galloyl-β-D-glucose, 230.76μg/ml of terminalic acid, 35.7μg/ml of urolithin M5, 183.6μg/ml of terminalic acid, 186.4μg/ml of terminalic acid and 175.32μg/ml of ellagic acid; a series of reference solutions of different concentrations were obtained by stepwise dilution.

Step c: The mixed reference solution and the test solution were measured 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:

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

Column temperature: 30°C;

Injection volume: 2 μL;

Flow rate: 0.3ml/min;

Mobile phase A was 0.10% v/v formic acid in water, mobile phase B was methanol, and linear gradient elution was performed. The elution program was:

The detection wavelength is 270nm;

The mass spectrometry conditions were as follows: data were collected in negative ion mode, and the acquisition mode was Auto MS/MS. The ion source parameters were set as follows: gas temperature was 200°C; drying gas flow rate was 12 L/min; nebulizer pressure was 40 psi; sheath gas temperature was 350°C; nozzle voltage was -1.5 kV; capillary voltage was -4.0 kV; fragmentor was 390 V; the mass-to-charge ratio (m/z) range scanned by the TOF analyzer was 100–1500 for MS ¹ and 50–1500 for MS ^2. The top three ions in the MS ¹ spectrum were automatically selected by collision-induced dissociation to trigger MS/MS fragmentation, and the collision energy was 30 V. Data acquisition was implemented on the Agilent MassHunter Workstation DataAcquisition software.

Test Example 1

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

(1) Study on linear relationship and detection limit and quantification limit

A series of mixed reference solutions were prepared according to the method of step b in Example 1, and the peak area was determined by ultra-high performance liquid chromatography. The chromatographic conditions were the same as step c in Example 1. The samples were injected twice in parallel, and the concentration of each compound reference substance was taken as the abscissa x (μg/ml), and the corresponding reference substance peak area was taken as the ordinate y to draw a regression equation; the detection limit and quantification limit of chebulic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic acid, chebulic acid, ellagic acid were determined with a signal-to-noise ratio S/N=3 as the detection limit (LOD) and S/N=10 as the quantification limit (LOQ). The results are shown in Table 2.

As shown in Table 2, each component has a good linear relationship within its respective concentration range (r ² ≥ 0.999), the concentration range of the detection limit is 0.0998–0.9779 μg/ml, and the concentration range of the quantification limit is 0.2559–1.956 μg/ml, indicating that the method for determining the contents of multiple components in Terminalia chebula provided by the present invention has high sensitivity.

(2) Precision test

(a) Intra-day precision test

The same sample solution was accurately pipetted and injected 6 times, and the peak area was determined by ultra-high performance liquid chromatography. The chromatographic conditions were the same as those in step c of Example 1. The RSD values were calculated based on the peak areas, and the results are shown in Table 3.

Table 3 Intra-day precision test results (n=6)

As can be seen from Table 3, the intra-day precision RSD of the method for determining the contents of multiple components in Terminalia chebula provided in Example 1 is less than 1.0%.

(b) Inter-day precision test

For 3 consecutive days, the same sample solution was precisely pipetted every day, and the injection was repeated 6 times. The peak area was determined by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those in step c of Example 1. The results are shown in Table 4.

Table 4 Results of inter-day precision test (n=3)

As can be seen from Table 4, the inter-day precision RSD of the method for determining the contents of multiple components in Terminalia chebula provided in Example 1 is less than 2.7%, and the determination method has good precision.

(3) Repeatability test

According to the method of step a in Example 1, 6 test solutions of terminalia chebula powder were prepared in parallel, and the test solutions were determined by ultra-high performance liquid chromatography to calculate the content of each compound. The chromatographic conditions were the same as step c in Example 1. The contents of terminalia chebula acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, terminalia chebula, urolithin M5, terminalia chebula tannic acid, terminalia chebula tannic acid, and ellagic acid were calculated using a standard curve and the RSD value was calculated. The results are shown in Table 5.

As shown in Table 5, in this test, the RSDs of the compounds were all less than 3.0%, including 41.48 mg/g of terminalia acid, 12.65 mg/g of gallic acid, 2.530 mg/g of 4-galloylshikimic acid, 3.760 mg/g of 5-galloylshikimic acid, 9.692 mg/g of punicalagin (punicalagin A & B), 3.809 mg/g of corilagin, 3.467 mg/g of 1,3,6-tri-O-galloyl-β-D-glucose, 17.56 mg/g of terminalia chebula, 2.707 mg/g of urolithin M5, 9.985 mg/g of terminalia chebula tannic acid, 7.878 mg/g of terminalia chebula tannic acid and 28.76 mg/g of ellagic acid. This indicated that the detection method had good repeatability.

(4) Stability test

The test solution was stored at 10°C, and the same portion of the test solution was accurately drawn at 0, 2, 4, 6, 8, 10, and 12 hours, respectively, and the test solution was measured by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those in step c of Example 1. The RSD value was calculated based on the peak areas of chebulic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic acid, chebulic acid, ellagic acid. The results are shown in Table 6.

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

(5) Sample recovery test

Take about 0.1g of terminalia chebula powder, weigh it accurately, and add 16.6ml of mixed reference solution with the same mass as the components in the sample (terminal acid 300.4μg/ml, gallic acid 84.34μg/ml, 4-galloylshikimic acid 18.40μg/ml, 5-galloylshikimic acid 29.92μg/ml, punicalagin (punicalagin A&B) 72.86μg/ml, corilagin 32.76μg/ml, 1,3,6-tri-O-galloyl-β-D-glucose 20.50μg/ml, terminalia chebula nin 115.4μg/ml, urolithin M5 17.85μg/ml, terminalia chebula tannic acid 64.26μg/ml, terminalia chebula tannic acid 55.92μg/ml, ellagic acid 175.3μg/ml).

Six samples of terminalia chebula test solution were prepared, and each sample was measured twice in parallel. The test solution was measured by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those in step c of Example 1. The accuracy of the method was investigated based on the sample recovery rate and RSD value calculated according to the sample recovery rate formula.

The recovery rate formula is as follows:

The results are shown in Table 7.

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

It can be seen from Tables 2 to 7 that the method for determining the contents of multiple components in Terminalia chebula provided by Example 1 basically meets the requirements of methodological research and can be used for quantitative analysis of Terminalia chebula.

Example 2

In this example, the content of tannin components in different batches of Terminalia chebula was detected by the content determination method in Example 1. The steps were the same as in Example 1, and the results are shown in Table 8.

Example 3

In this example, the tannin components in the citrus fruit were detected using the content determination method in Example 1.

Step a, preparation of test solution: same as Example 1, except that the terminalia chebula powder is replaced with citronella powder;

Step b, preparation of reference solution: same as in Example 1;

Step c: Same as Example 1. The results are shown in Table 9.

The liquid chromatogram of each medicinal material test solution is shown in Figure 1, the TIC mass spectrum is shown in Figure 2, and the qualitative detection results are shown in Table 10.

Example 4

The embodiment of the present invention provides a method for constructing a characteristic fingerprint of Terminalia chebula, comprising the following steps:

Step 1, prepare a test solution of terminalia chebula powder and a mixed reference solution according to the preparation method of the test solution and the reference solution in Example 1;

Step 2: Same as step c in Example 1, record the characteristic fingerprint.

The data was imported into the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system for processing. The spectrum of Terminalia chebula HZ35 was used as the reference spectrum, and 12 main characteristic peaks with strong absorption signals, good separation and stability were selected as common peaks, as shown in Figure 3. The similarity between the Terminalia chebula fingerprint spectrum and the reference fingerprint spectrum was calculated. The results showed that the similarity between 35 batches of Terminalia chebula samples and the reference spectrum was greater than 0.66, indicating that the difference between Terminalia chebula batches was large. According to the determination results of the relative retention time of each compound in the fingerprint spectrum of 35 batches of Terminalia chebula medicinal materials, 12 common chromatographic peaks with large peak area, good peak shape and separation were selected as characteristic fingerprint peaks. The standard reference fingerprint spectrum was generated, with the H6 peak as the reference peak. The relative retention time and relative peak area of the 12 common chromatographic peaks are shown in Tables 11 and 12. By comparing with the chromatograms of reference substances, the characteristic fingerprint peaks were identified as chebulic acid (H1), gallic acid (H2), 5-galloylshikimic acid (H3), punicalagin A (H4), punicalagin B (H5), corilagin (H6), 1,3,6-tri-O-galloyl-β-D-glucose (H7), chebulin (H8), chebulic tannic acid (H9), chebulin tannic acid (H10), ellagic acid (H11), and 4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (H12).

Example 5

The embodiment of the present invention provides a method for constructing a characteristic fingerprint of Xiqing fruit, comprising the following steps:

Step 1: prepare a test solution of Xiqing fruit powder and a mixed reference solution according to the preparation method of the test solution and the reference solution in Example 1.

Step 2: Same as step c in Example 1, record the characteristic fingerprint.

The data was imported into the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system for processing. The spectrum of Xiqingguo XQG24 was used as the reference spectrum, and 11 main characteristic peaks with strong absorption signals, good separation and stability were selected as common peaks, as shown in Figure 4. The similarity between the UPLC spectrum of each sample and the reference fingerprint spectrum was calculated. The results showed that the similarity between the 28 batches of Xiqingguo samples and the reference spectrum was greater than 0.86, and the similarity was good. According to the determination results of the relative retention time of each compound in the fingerprint spectrum of 28 batches of Xiqingguo medicinal materials, 11 common chromatographic peaks with large peak area, good peak shape and separation were selected as characteristic fingerprint peaks. The standard reference fingerprint spectrum was generated, with the X5 peak (corilagin) as the reference peak. The relative retention time and relative peak area of the 11 common chromatographic peaks are shown in Tables 13 and 14. By comparing with the chromatogram of the reference substance, the characteristic fingerprint peaks were identified as chebulic acid (X1), gallic acid (X2), punicalagin A (X3), punicalagin B (X4), corilagin (X5), 1,3,6-tri-O-galloyl-β-D-glucose (X6), chebulic acid (X7), urolithin M5 (X8), chebulic acid (X9), chebulic acid (X10), and ellagic acid (X11).

The peak of corilagin is used as the reference peak (X5), and the relative retention time of each common peak and the reference peak is within the range of ±5% of the specified value; the specified values are: X1 peak (chebulic acid) is 0.2232-0.2466, X2 peak (gallic acid) is 0.4239-0.4686, X3 peak (punicalagin A) is 0.7401-0.818, X4 peak (punicalagin B) is 0.8442-0.933, X5 peak (corilagin) is 1 .000, X6 peak (1,3,6-tri-O-galloyl-β-D-glucose) was 1.057-1.168, X7 peak (chebula tannin) was 1.167-1.290, X8 peak (urolithin M5) was 1.201-1.327, X9 peak (chebula tannic acid) was 1.307-1.444, H10 peak (chebula tannic acid) was 1.503-1.662, and X11 peak (ellagic acid) was 1.533-1.694.

Example 6

The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution: same as in Example 1;

Step b, preparation of reference solution: same as in Example 1;

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

Example 7

The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution: same as in Example 1;

Step b, preparation of reference solution: same as in Example 1;

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

Example 8

The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution: same as in Example 1;

Step b, preparation of reference solution: same as in Example 1;

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

The liquid chromatograms of the test solutions in Examples 1, 6, 7, and 8 are shown in FIG5 . The peaks of the 13 tannin compounds can be separated at a column temperature of 30 to 60° C., and the peak separation of the 13 compounds is better at a column temperature of 30° C.

Example 9

The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of the test solution: take 0.2g of Terminalia chebula powder, accurately weigh it, place it in a 25ml volumetric flask, add appropriate amount of methanol, ultrasonically extract at 30℃ for 20min, leave it at room temperature, dilute to the scale with methanol, shake well, take appropriate amount, centrifuge at 13700rpm for 10min, and 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.

Step b, preparation of reference solution: same as in Example 1.

Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.

The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 15.

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

As can be seen from Table 15, when methanol is used as the extraction solvent, the peak area of tannin components is the highest and the extraction effect is the best.

Example 10

The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution: accurately weigh 1, 0.2, 0.2, 0.1g of Terminalia chebula powder, and place them in 50ml, 25ml, 50ml, and 50ml volumetric flasks, respectively, accurately add appropriate amount of methanol, ultrasonically extract at 30℃ for 20min, leave at room temperature, dilute to the scale with methanol, shake well to obtain solutions with solid-liquid ratio of 1:50, 1:125, 1:250, and 1:500, respectively, take appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution.

Step b, preparation of reference solution: same as in Example 1.

Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.

The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 16.

Table 16 Peak areas of tannin components in the test solution obtained under different material-liquid ratios

As can be seen from Table 16, when the solid-liquid ratio is 1:250 and 1:500, the peak area of tannin components is relatively high, the extraction effect is better, and the preferred solid-liquid ratio is 1:250.

Embodiment 11

The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution: take 0.2g of Terminalia chebula powder, accurately weigh it, put it in a 50ml volumetric flask, add an appropriate amount of methanol, ultrasonically extract at 30℃ for 10, 20, and 30min respectively, after leaving it at room temperature, add methanol to the scale, shake well, take an appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution.

Step b, preparation of reference solution: same as in Example 1.

Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.

The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 17.

Table 17 Peak areas of tannin components in the test solution obtained at different ultrasonic times

As can be seen from Table 17, when the ultrasonic time is 20 min and 30 min, the peak area of tannin components is higher and the extraction effect is better, so the ultrasonic time of 20 min is selected.

Example 12

The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of test solution: take 0.2g of Terminalia chebula powder, accurately weigh it, put it in a 50ml volumetric flask, add appropriate amount of methanol, ultrasonically extract at 30, 40, and 50℃ for 20min respectively, after leaving it at room temperature, dilute to the scale with methanol, shake well, take appropriate amount respectively, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution.

Step b, preparation of reference solution: same as in Example 1.

Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.

The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 18.

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

As can be seen from Table 18, when the ultrasonic temperature is 30°C, the peak area of tannin components is the highest and the extraction effect is optimal.

Comparative Example 13

The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:

Step a, preparation of the test solution: the same as the preparation of the reference solution in Example 1;

Step b: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the above-mentioned test solution, the chromatographic conditions are:

Chromatographic column: ZORBAX SB-C18 (4.6x 100mm, 1.8μm), ACQUITY UPLC HSS T3 (2.1x100mm, 1.8μm) or Cosmosil PBr Packed Column (2.1x 100mm, 2.6μm);

Column temperature: 30°C

Injection volume: 2 μL;

Flow rate: 0.3ml/min;

Mobile phase A was 0.1% v/v formic acid in water, mobile phase B was methanol, and linear gradient elution was performed. The elution program was:

The chromatogram obtained when the chromatographic column is ZORBAX SB-C18 is shown in Figure 6A; the chromatogram when the chromatographic column is ACQUITYUPLC HSS T3 is shown in Figure 6B; and the chromatogram when the chromatographic column is Cosmosil PBr Packed Column is shown in Figure 6C.

As shown in Figure 6, the Cosmosil PBr Packed Column is more suitable for the separation of tannin compounds (similar polarity) in Terminalia chebula and has a better separation degree.

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 in the protection scope of the present invention.

Claims (10)

1. a method for assaying the content of multi-components in the fruit of Myrobalan, it is characterized in that, the fruit of Myrobalan is extracted with aqueous methanol, and the composition to be measured is carried out to assay with ultra-high performance liquid chromatography, and the composition to be measured includes the fruit of Myrobalan Acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A&B), corilagin, 1,3,6-tri-O-galloyl-β-D- Glucose, chebulin, urolithin M5, chebulin, chebulin and ellagic acid; the chromatographic conditions of the ultra-high performance liquid chromatography include: Chromatographic column: Pentabromophenyl HPLC column; Mobile phase: mobile phase A is 0.095%～0.105% v/v formic acid aqueous solution, mobile phase B is methanol, gradient elution, the program of described gradient elution is as follows: Flow rate: 0.28～0.32ml/min; The detection wavelength is: 250-290nm. 2. The assay method according to claim 1, characterized in that, the volume percent concentration of the aqueous methanol solution is 40% to 100%; and/or The extraction method is ultrasonic extraction, the extraction temperature is 30-50° C., and the extraction time is 10-30 minutes. 3. The content determination method according to claim 1, characterized in that, the Pentabromophenyl HPLC column is a Cosmosil PBr Packed Column with a specification of 2.1×100mm and 2.6 μm. 4. The assay method according to claim 1, characterized in that mobile phase A is 0.10% v/v formic acid aqueous solution; and/or. The flow rate is 0.3ml/min. 5. The content determination method according to claim 1, characterized in that, the chromatographic conditions further include a column temperature of 30-60°C. 6. The assay method according to any one of claims 1 to 5, characterized in that it comprises the following steps: Step a, extract Myrobalan with methanol aqueous solution, make need testing solution; Step b, prepare the reference substance solution of the component to be tested; Step c, using the ultra-high performance liquid chromatography to measure the reference substance solution and the test solution, and use the standard curve method to calculate the content of the target index components in the test product. 7. a method for constructing characteristic fingerprints of Myrobalan chebula, is characterized in that, comprises the following steps: S1. Preparation of reference substance solution of index components; said index components include chebulinic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin , 1,3,6-tri-O-galloyl-β-D-glucose, chebulinin, urolithin M5, chebulin tannic acid, chebulin tannic acid and ellagic acid; S2, get different batches of Myrobalan, extract with methanol respectively, obtain the need testing solution of different batches of Myrobalan, use the ultra-high performance liquid chromatography in the above-mentioned assay method for the reference substance solution and the test sample The solution is measured, the characteristic fingerprints are recorded, and all the characteristic fingerprints are imported into the similarity evaluation system software of traditional Chinese medicine chromatographic fingerprints, and a batch of Myrobalan chebulae is used as a reference spectrum, and the selected absorption signal is stronger, and the separation degree and stability are relatively high. Good peaks are used as characteristic peaks, and the relative retention time and relative peak area of each characteristic peak are calculated. 8. A characteristic fingerprint of Myrobalan, characterized in that it contains 12 characteristic fingerprint peaks: followed by chebulinic acid, gallic acid, 5-galloylshikimic acid, punicalagin A, punicalagin B, and collella Jing, 1,3,6-tri-O-galloyl-β-D-glucose, chebulinin, urolithin M5, chebulin tannic acid, chebulin tannic acid and ellagic acid. 9. chebula characteristic fingerprint collection according to claim 8, it is characterized in that, with the peak of Corilagin as reference peak, the relative retention time of each common peak and reference peak is within the scope of ± 5% of specified value _The specified values are: _{0.2220-0.2453} for H1 peak, 0.4239-0.4685 _for H2 peak, _{0.6435-0.7112} for H3 peak, 0.7405-0.8185 for H4 peak, 0.8456-0.9346 for H5 peak, 1.000 _for H6 peak, 1.056-0.056 for H7 _peak 1.167, H8 peak is 1.167 _– 1.290, H9 peak is 1.306 _– 1.444, H10 peak is 1.501 _– 1.659, H11 peak is 1.532 _– 1.693, H12 peak is 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 of tannin components in green fruit and the construction of characteristic fingerprints.