CN111830160B

CN111830160B - Method for detecting fingerprint spectrum of Qijudihuang pills and application thereof

Info

Publication number: CN111830160B
Application number: CN202010702746.2A
Authority: CN
Inventors: 沈丹萍; 张正光; 詹常森; 姜鹏; 温方方
Original assignee: Shanghai Hutchison Pharmaceuticals Ltd
Current assignee: Shanghai Hutchison Pharmaceuticals Ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2022-06-14
Anticipated expiration: 2040-07-21
Also published as: CN111830160A

Abstract

The invention provides a method for detecting a fingerprint spectrum of Qijudihuang pills. The invention also provides application of the detection method of the fingerprint spectrum of the Qijudihuang pill in quality detection of components in the Qijudihuang pill and a quality detection method thereof. The invention further provides a method for measuring the content of 9 components in the Qijudihuang pill. The invention further provides a screening method of the fingerprint spectrums of the multiple medicinal materials in the Qijudihuang pill. The invention provides a detection method of fingerprint of Qiju Dihuang Wan and application thereof, which establishes a high-performance liquid fingerprint of the Qiju Dihuang Wan, can carry out quantitative analysis on 9 chemical components in the Qiju Dihuang Wan, and can carry out attribution confirmation on chromatographic peaks of each single medicinal material in the Qiju Dihuang Wan, can effectively monitor from a source of raw materials, strictly control the quality of the raw medicinal materials, and ensure that the amount of the effective components in the Qiju Dihuang Wan is relatively stable, thereby ensuring the safety and effectiveness of clinical medication.

Description

Method for detecting fingerprint spectrum of Qijudihuang pills and application thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicine component detection, relates to a detection method of a fingerprint of Qijudihuang pills and application thereof, and particularly relates to a detection method of a fingerprint of Qijudihuang pills and application thereof in quality detection.

Background

The Qiju Dihuang Wan is a medicine for nourishing kidney and liver, and is clinically used for treating yin deficiency of liver and kidney, dizziness and tinnitus, photophobia, epiphora induced by wind and blurred vision. Although the pill of medlar, chrysanthemum and rehmannia reveals that the medicinal materials comprise 8 medicines, namely medlar, prepared rehmannia root, tree peony bark, tuckahoe, chrysanthemum, wine dogwood fruit, yam and rhizoma alismatis, the contained chemical components are extremely complex. In the ' pharmacopoeia of the people's republic of China ' 2020 edition, only the characters, microscopic identification and general inspection of pill items of the pill of Chinese wolfberry, chrysanthemum and rehmannia are specified, the wolfberry is identified by a thin layer, and quantitative requirements are made on noroside, loganin and paeonol, so that the overall quality of the pill of Chinese wolfberry, chrysanthemum and rehmannia can not be comprehensively and accurately evaluated.

The traditional Chinese medicine fingerprint has the characteristics of integrity, macroscopicity, fuzzy analysis and the like, can comprehensively reflect the types and the quantity of chemical components contained in the traditional Chinese medicine by describing the integral characteristics of the traditional Chinese medicine and adopting a proper fuzzy processing mode, and achieves the aim of integral quality control, thereby becoming an effective means for the quality control of the traditional Chinese medicine and being widely applied to the field of the quality monitoring and evaluation of the traditional Chinese medicine. The chromatographic fingerprint analysis can visualize the overall characteristics of various chemical components contained in the traditional Chinese medicine, thereby revealing the quality problem which is difficult to find by the conventional inspection. The fingerprint spectrum is a modern Chinese medicine quality control method from the perspective of 'full components' according to the characteristics of the overall comprehensive action of multiple components and multiple target points of the Chinese medicine, and the quality control of the non-single component medicine is more comprehensive. Currently, systematic qualitative and quantitative research on the pill of Chinese wolfberry, chrysanthemum and rehmannia is lacked. Therefore, it is necessary to establish a fingerprint spectrum and multi-component content measurement of the pill of Chinese wolfberry and rehmanniae, and to complete qualitative and quantitative analysis of the components in the pill of Chinese wolfberry and rehmanniae, so as to perform effective quality control on the pill of Chinese wolfberry and rehmanniae.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a detection method of a fingerprint of a pill of Chinese wolfberry, chrysanthemum and rehmannia and application thereof, wherein the pretreatment of optimized conditions and a high performance liquid chromatography method are adopted to establish the high performance liquid chromatography fingerprint of the pill of Chinese wolfberry, chrysanthemum and rehmannia, and the contributions of different chemical components to a fingerprint system of the pill of Chinese wolfberry, chrysanthemum and rehmannia are highlighted from different sides, so that the detection of the whole chemical components of the pill of Chinese wolfberry, chrysanthemum and rehmannia is realized, the current situation of each component in the pill of Chinese wolfberry, chrysanthemum and rehmannia is comprehensively reflected, and a reference basis is provided for the overall control and evaluation of the quality of the pill of Chinese wolfberry, chrysanthemum and rehmannia.

In order to achieve the above objects and other related objects, a first aspect of the present invention provides a method for detecting a fingerprint of qijudihuang pills, comprising the steps of:

1) preparation of a test solution: adding methanol water solution into the pill sample of Chinese wolfberry, chrysanthemum and rehmannia, carrying out ultrasonic extraction, and filtering supernatant to obtain a test solution;

2) preparation of control solutions: adding methanol water solution into a reference substance of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B for dissolving to prepare a reference substance solution;

3) and (3) determination: respectively measuring the test solution and the reference solution by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions to obtain a fingerprint of the test solution and a fingerprint of the reference solution, comparing the fingerprint of the test solution with the fingerprint of the reference solution, and performing attribution positioning on index components in the fingerprint of the test solution, thereby obtaining the fingerprint of the Qiju-dihuang pill.

Preferably, in the step 1), the sample of the pill of engelhardtia is a powder sample obtained by pulverizing the pill of engelhardtia.

Preferably, in step 1), the ratio of the weight (g) of the added sample of qiju dihuang pills to the volume (mL) of the added methanol aqueous solution is 4: 120-130. More preferably, the ratio of the weight (g) of the added sample of qiju dihuang pills to the volume (mL) of the added methanol aqueous solution is 4: 125.

preferably, in the step 1), the sample of the pill of Chinese wolfberry and rehmannia and the methanol water solution are weighed and then need to be weighed.

Preferably, in the step 1), the ultrasonic extraction time is 20-40 min. More preferably, the ultrasound extraction time is 30 min.

Preferably, in the step 1), the power of the ultrasonic extraction is 200-300W, and the frequency of the ultrasonic extraction is 30-50 kHz. More preferably, the power of the ultrasonic extraction is 250W, and the frequency of the ultrasonic extraction is 40 kHz.

Preferably, in step 1), the sample of Qiju Dihuang Wan is precisely weighed after being added with the methanol water solution.

Preferably, in step 1), the ultrasonic extraction is followed by cooling, reweighing, and weight loss compensation with aqueous methanol.

Preferably, in the step 1), the filtration mode is a filter membrane filtration mode. More preferably, the pore size of the filter is 0.22 μm.

Preferably, in step 1) or 2), the methanol aqueous solution is methanol aqueous solution with volume percentage concentration of more than or equal to 50%.

More preferably, the methanol aqueous solution is 80% methanol aqueous solution by volume.

Preferably, in the step 2), the CAS number of the 5-hydroxymethylfurfural is 67-47-0, the CAS number of the morroniside is 25406-64-8, the CAS number of the chlorogenic acid is 327-97-9, the CAS number of the loganin is 18524-94-2, the CAS number of the paeoniflorin is 23180-57-6, the CAS number of the luteolin is 5373-11-5, the CAS number of the 3, 5-O-dicaffeoylquinic acid is 2450-53-5, the CAS number of the paeonol is 552-41-0, and the CAS number of the 23-acetyl alisol B is 26575-95-1.

Preferably, in step 2), the content ranges of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B in the control solution are all 0.5-1000 μ g/mL.

More preferably, the content range of each component in the control solution is as follows: 1.43-286.00 mu g/mL of 5-hydroxymethylfurfural, 1.80-359.21 mu g/mL of morroniside, 0.88-88.28 mu g/mL of chlorogenic acid, 2.16-431.21 mu g/mL of loganin, 2.15-430.61 mu g/mL of paeoniflorin, 1.38-138.38 mu g/mL of luteolin, 7.57-378.71 mu g/mL of 3, 5-O-dicaffeoylquinic acid, 9.76-976.40 mu g/mL of paeonol and 1.14-114.40 mu g/mL of 23-acetyl alisol B.

Most preferably, the content range of each component in the control solution is as follows: 28.60 mu g/mL of 5-hydroxymethylfurfural, 35.92 mu g/mL of morroniside, 17.66 mu g/mL of chlorogenic acid, 43.12 mu g/mL of loganin, 43.06 mu g/mL of paeoniflorin, 27.68 mu g/mL of luteolin, 37.87 mu g/mL of 3, 5-O-dicaffeoylquinic acid, 97.64 mu g/mL of paeonol and 22.88 mu g/mL of 23-acetyl alisol B.

Preferably, in the step 2), the reference solution is prepared by stepwise dilution. When in use, the reference substance solution is shaken up and then filtered, and the subsequent filtrate is taken.

More preferably, the stepwise dilution of the control solution comprises the steps of:

A1) adding methanol water solution into a reference substance of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B for dilution and volume fixing to prepare a reference substance stock solution;

A2) adding methanol water solution into the reference substance stock solution for dilution and constant volume to prepare the required reference substance solution.

Further preferably, in step a1), the concentrations of the components in the control stock solution are respectively: 0.286mg/mL of 5-hydroxymethylfurfural, 0.359mg/mL of morroniside, 0.177mg/mL of chlorogenic acid, 0.431mg/mL of loganin, 0.431mg/mL of paeoniflorin, 0.277mg/mL of luteolin, 0.379mg/mL of 3, 5-O-dicaffeoylquinic acid, 0.976mg/mL of paeonol and 0.229mg/mL of 23-acetyl alisol B.

Further preferably, in step a1), the control stock solution is stored in a refrigerator at 4 ℃ protected from light.

Further preferably, in step A1) or A2), the methanol aqueous solution has a volume percentage concentration of more than or equal to 50%. Most preferably, the aqueous methanol solution is 80% by volume aqueous methanol solution.

Preferably, in the step 3), the fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peak in the fingerprint of the test solution is identified through the relative retention time according to the known characteristic peak in the fingerprint of the reference solution, so as to perform attribution positioning on the index component in the fingerprint of the test solution.

Preferably, in the step 3), the chromatographic column in the high performance liquid chromatography is a C18 chromatographic column. More preferably, the column in the high performance liquid chromatography is an Agilent 5TC-C18 column (4.6 mm. times.250 mm, 5 μm).

Preferably, in step 3), the column temperature in the high performance liquid chromatography is 25-35 ℃. More preferably, the column temperature in the high performance liquid chromatography is 30 ℃.

Preferably, in the step 3), the sample amount in the high performance liquid chromatography is 1 to 20 μ L. More preferably, the amount of sample in the high performance liquid chromatography is 10. mu.L.

Preferably, in the step 3), the flow rate in the high performance liquid chromatography is 0.8-1.0 mL/min; the mobile phase is acetonitrile-0.05-0.2% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.2% phosphoric acid water solution; the analysis time is 85 min; gradient elution.

The 0.05-0.2% phosphoric acid aqueous solution is 0.05-0.2% phosphoric acid aqueous solution by volume percentage.

More preferably, the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, wherein phase a is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution.

More preferably, as shown in table 1, the specific procedure of the gradient elution is:

0-12min, phase A: the volume ratio of the phase B is 5: 95-9: 91, flow rate of 1.0 mL/min;

12-31min, phase A: the volume ratio of the phase B is 9: 91-19: 81, the flow rate is 1.0 mL/min;

31-35min, phase A: the volume ratio of the phase B is 19: 81-19: 81, the flow rate is 1.0 mL/min;

35-50min, phase A: the volume ratio of the phase B is 19: 81-27: 73, the flow rate is 1.0 mL/min;

50-60min, phase A: the volume ratio of the phase B is 27: 73-69: 31, the flow rate is 1.0 mL/min;

60-65min, phase A: the volume ratio of the phase B is 69: 31-81: 19, the flow rate is 1.0 mL/min;

65-70min, phase A: the volume ratio of the phase B is 81: 19-95: 5, the flow rate is 0.8 mL/min;

70-85min, phase A: the volume ratio of the phase B is 95: 5-95: 5, the flow rate was 0.8 mL/min.

TABLE 1 gradient elution procedure

Time/min	Phase A: methanol/% of	Phase B: 0.1% phosphoric acid/%)	Flow rate mL/min
				0	5	95	1.0
12	9	91	1.0
				31	19	81	1.0
35	19	81	1.0
				50	27	73	1.0
60	69	31	1.0
				65	81	19	1.0
70	95	5	0.8
				85	95	5	0.8

Preferably, in the step 3), the scanning wavelength in the high performance liquid chromatography is 200-350 nm. More preferably, the scanning wavelength in the high performance liquid chromatography is 210-334 nm.

More preferably, as shown in table 2, the specific procedure of the change of the scanning wavelength with the retention time is as follows:

scanning wavelength of 274nm for 0-17 min;

17-17.01min, the scanning wavelength is changed from 274nm to 240 nm;

17.01-34min, and the scanning wavelength is 240 nm;

34-34.01min, and changing the scanning wavelength from 240nm to 334 nm;

34.01-68min, and the scanning wavelength is 334 nm;

68-68.01min, and changing the scanning wavelength from 334nm to 210 nm;

68.01-85min, and the scanning wavelength is 210 nm.

TABLE 2 specific procedure for scanning wavelength as a function of retention time

Retention time/min	Wavelength nm
			0	274
17	274
		17.01	240
34	240
		34.01	334
68	334
		68.01	210
85	210

The second aspect of the invention provides an application of a detection method of a fingerprint spectrum of Qijudihuang pills in quality detection of components in the Qijudihuang pills.

The third aspect of the invention provides a quality detection method of Qijudihuang pills, which comprises the steps of obtaining a fingerprint of the Qiju pill by adopting the detection method of the fingerprint of the Qiju pill, and comparing the similarity of the obtained fingerprint of the Qiju pill with a standard fingerprint of the Qiju pill obtained under the same fingerprint detection condition.

Preferably, when the similarity of the measured fingerprint of the pill of Chinese wolfberry and rehmanniae radix is compared with the standard fingerprint of the pill of Chinese wolfberry and rehmanniae radix, the similarity is compared by using software 2009 (evaluation system for chromatographic fingerprint of traditional Chinese medicine) issued by the committee of national pharmacopoeia. More preferably, the similarity between the fingerprint of the pill of Chinese wolfberry and rehmannia measured by the method and the standard fingerprint of the pill of Chinese wolfberry and rehmannia is more than or equal to 0.95.

Preferably, the standard fingerprint of the pill of Chinese wolfberry and rehmannia is obtained by the same conditions as the detection method of the fingerprint of the pill of Chinese wolfberry and rehmannia, and comprises 15 common fingerprint peaks, wherein the 13 peak is used as a positioning peak (S peak, retention time is 1.000), and the relative retention times of the other 14 common fingerprint peaks are sequentially 1 peak (0.133 +/-0.002), 2 peak (0.168 +/-0.002), 3 peak (0.354 +/-0.002), 4 peak (0.402 +/-0.003), 5 peak (0.470 +/-0.002), 6 peak (0.531 +/-0.002), 7 peak (0.693 +/-0.003), 8 peak (0.712 +/-0.004), 9 peak (0.776 +/-0.003), 10 peak (0.6 +/-0.003), 11 peak (0.829 +/-0.003), 12 peak (0.002), 14.921 +/-0.002), and 1.15 peak (15.921 +/-0.002).

More preferably, the standard fingerprint of the pill of Chinese wolfberry and rehmannia comprises 15 common fingerprint peaks, wherein the 13 peak is taken as a positioning peak (S peak, retention time is 1.000), and the relative retention time of the other 14 common fingerprint peaks is sequentially No. 1 peak (0.133 +/-0.001), No. 2 peak (0.168 +/-0.001), No. 3 peak (0.354 +/-0.001), No. 4 peak (0.402 +/-0.002), No. 5 peak (0.470 +/-0.001), No. 6 peak (0.79 +/-0.001), No. 7 peak (0.693 +/-0.002), No. 8 peak (0.712 +/-0.003), No. 9 peak (0.470 +/-0.002), No. 10 peak (0.796 +/-0.002), No. 11 peak (0.829 +/-0.002), No. 12 peak (0.921 +/-0.001), No. 14 peak (1.206 +/-0.001) and No. 15.001 +/-0.002).

Most preferably, the standard fingerprint of the pill of Chinese wolfberry and rehmannia comprises 15 common fingerprint peaks, wherein the 13 peak is taken as a positioning peak (S peak, retention time is 1.000), and the relative retention time of the other 14 common fingerprint peaks is sequentially No. 1 peak (0.133), No. 2 peak (0.168), No. 3 peak (0.354), No. 4 peak (0.402), No. 5 peak (0.470), No. 6 peak (0.531), No. 7 peak (0.693), No. 8 peak (0.712), No. 9 peak (0.776), No. 10 peak (0.796), No. 11 peak (0.829), No. 12 peak (0.921), No. 14 peak (1.206) and No. 15 peak (1.245). The specific data of the standard fingerprint of QIJUDIHUANG pill is shown in figure 1.

More preferably, as shown in fig. 4, the standard fingerprint of the pill of chinese wolfberry and rehmannia is compared with the fingerprint of the control solution, and the peak 1 is located and determined to be the fingerprint of 5-hydroxymethylfurfural, the peak 2 is the fingerprint of morroniside, the peak 3 is the fingerprint of chlorogenic acid, the peak 4 is the fingerprint of loganin, the peak 5 is the fingerprint of paeoniflorin, the peak 6 is the fingerprint of luteolin, the peak 7 is the fingerprint of 3, 5-O-dicaffeoylquinic acid, the peak 8 is the fingerprint of paeonol, and the peak 9 is the fingerprint of 23-acetylalisol B.

The fourth aspect of the invention provides a method for measuring the content of 9 components in Qijudihuang pills, which comprises the following steps:

a) preparing a test solution: the method is the same as the step 1) of the detection method of the fingerprint spectrum of the Qijudihuang pill;

b) preparation of control solutions: the detection method is the same as the step 2) of the fingerprint spectrum detection method of the Qijudihuang pill;

c) and (3) determination: respectively measuring the test solution in the step a) and the reference solution in the step b) by adopting a high performance liquid chromatography with the same chromatographic conditions as those in the detection method of the fingerprint of the Qiju-Dihuang pill, and calculating the content of 9 components in the test solution by adopting an external standard method.

Preferably, the external standard method is selected from one of an external standard point method or a standard curve method.

The external standard point method is as follows: transferring a certain volume of the reference substance in the step b) to prepare a reference substance solution with a certain concentration, analyzing by adopting a high performance liquid chromatograph to obtain a linear relation between the content of the 9 components in the reference substance solution and the peak area, drawing corresponding working curves according to the peak area of each component chromatogram corresponding to the corresponding content, and calculating to obtain a regression equation of each working curve. And detecting the sample solution by using a high performance liquid chromatograph, and substituting the chromatographic peak areas of the 9 components in the obtained sample solution into the regression equation of each working curve respectively to obtain the content of the corresponding component.

The standard curve method is as follows: respectively transferring a series of reference substances in the step b) with different volumes to prepare a series of reference substance solutions with different concentrations, adopting a high performance liquid chromatograph to perform sample injection analysis to obtain a linear relation between the content of 9 components in the reference substance solution and the peak area, drawing corresponding standard working curves according to the peak area of each component chromatographic spectrum corresponding to the corresponding content, and calculating to obtain a regression equation of each standard working curve. And detecting the sample solution by using a high performance liquid chromatograph, and substituting the chromatographic peak areas of the 9 components in the obtained sample solution into the regression equation of each standard working curve respectively to obtain the content of the corresponding component.

The fifth aspect of the invention provides a method for screening fingerprint spectrums of multiple medicinal materials in Qijudihuang pills, which comprises the following steps:

A) preparing a sample solution of a single medicinal material: preparing one or more of 8 medicinal material samples of fructus Lycii, radix rehmanniae Preparata, cortex moutan, Poria, flos Chrysanthemi, Corni fructus with wine, rhizoma Dioscoreae, and Alismatis rhizoma in QIJUDIHUANG pill according to step 1) of detection method of fingerprint of QIJUDIHUANG pill to obtain at least one single medicinal material sample solution;

B) and (3) determination: determining the sample solution of the single medicinal material by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions as the step 3) of the fingerprint detection method of the Qijudihuang pill to obtain the fingerprint of the sample solution of the single medicinal material;

C) obtaining a standard fingerprint: obtaining a standard fingerprint spectrum of the Qijudihuang pill by adopting the same steps as the detection method of the fingerprint spectrum of the Qijudihuang pill;

D) and (3) quality detection: comparing the fingerprint of the single medicinal material sample solution with the standard fingerprint of the Qijudihuang pill, and identifying the corresponding characteristic peak of the single medicinal material sample solution in the standard fingerprint of the Qijudihuang pill through relative retention time, thereby performing attribution positioning on the characteristic peak in the fingerprint of the single medicinal material sample solution.

Preferably, in the step A), the amount of the aqueous methanol solution is 1g of Lycium barbarum (Lycii Fructus), 1g of Rehmanniae Radix Preparata (Rehmanniae Radix Praeparata), 1g of Moutan Cortex (Moutan Cortex), 1g of Poria (Poria), 1g of Chrysanthemum morifolium (Chrysanthemum Flos), 1g of Cornus officinalis (Wine-prepared Corni Fructus), 1g of Dioscorea opposita (Dioscorea rhizome) and 1g of Alisma orientale (Alisma Rhizoma) per 200-300 mL.

More preferably, the methanol aqueous solution contains 1g of medlar, 1g of prepared rehmannia root, 1g of moutan bark, 1g of tuckahoe, 1g of chrysanthemum, 1g of dogwood fruit, 1g of yam and 1g of rhizoma alismatis per 250 mL.

Preferably, in the step D), the attribution of the characteristic peaks of the measured fingerprint spectrum of the single medicinal material sample solution and the standard fingerprint spectrum of the pill of Chinese wolfberry and rehmannia is positioned, the analysis and the processing are performed by using software 2009, published by the national pharmacopoeia committee, of the similarity evaluation system of traditional Chinese medicine chromatography fingerprint spectrum, and the attribution of the characteristic peaks of each single medicinal material of the pill of Chinese wolfberry and rehmannia is confirmed by the relative retention time of each characteristic peak on the standard fingerprint spectrum of the pill of Chinese wolfberry and chrysanthemum and rehmannia.

Preferably, in the step D), the fingerprint of the prepared rehmannia root sample solution in the single herb sample solution comprises 1 common fingerprint peak, and the 1 common fingerprint peak is peak No. 2. Specific results are shown in Table 3.

Preferably, in the step D), the fingerprint of the moutan bark sample solution in the single herb sample solution includes 2 common fingerprint peaks, and the 2 common fingerprint peaks are peak 6 and peak 13. The specific results are shown in Table 3.

Preferably, in the step D), the fingerprint of the chrysanthemum sample solution includes 7 common fingerprint peaks, and the 7 common fingerprint peaks are No. 4 peak, No. 7 peak, No. 8 peak, No. 9 peak, No. 10 peak, No. 11 peak and No. 12 peak. The specific results are shown in Table 3.

Preferably, in the step D), the fingerprint of the sample solution of dogwood fruit wine comprises 4 common fingerprint peaks, wherein the 4 common fingerprint peaks are peak 1, peak 2, peak 3 and peak 5. The specific results are shown in Table 3.

Preferably, in the step D), the fingerprint of the alisma orientale sample solution in the single medicinal material sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are peak 14 and peak 15. The specific results are shown in Table 3.

Preferably, in the step D), the fingerprint spectrums of the wolfberry fruit sample solution, the poria cocos sample solution and the yam sample solution in the single medicinal material sample solution do not include a common fingerprint peak. The specific results are shown in Table 3.

In the common fingerprint peaks, after comparison of a mixed reference substance containing 9 components in fig. 3, the peak 2 is determined to be 5-hydroxymethylfurfural, the peak 3 is morroniside, the peak 4 is chlorogenic acid, the peak 5 is loganin, the peak 6 is paeoniflorin, the peak 7 is luteolin, the peak 9 is 3, 5-O-dicaffeoylquinic acid, the peak 13 is paeonol, and the peak 15 is 23-acetylalisol B.

TABLE 3 common fingerprint attribution of each single medicinal material in Qijudihuang pill

Medicinal materials	Number of peaks (number)	Common fingerprint peak number
			Chinese wolfberry fruit	0	—
Prepared rehmannia root	1	2
			Cortex moutan	2	6、13
Poria cocos (Schw.) wolf	0	—
			(Chrysanthemum)	7	4、7、8、9、10、11、12
Wine cornus pulp	4	1、2、3、5
			Chinese yam	0	—
Rhizoma alismatis	2	14、15

The water used in the invention is pure water.

As mentioned above, the detection method of the fingerprint of the Qijudihuang pill and the application thereof provided by the invention adopt the pretreatment of optimized reaction conditions and the high performance liquid chromatography method to establish the high performance liquid chromatography fingerprint of the Qijudihuang pill, and the contributions of different chemical components to the fingerprint system of the Qijudihuang pill are highlighted and reflected from different side faces, thereby realizing the detection of the full-formula chemical components of the Qijudihuang pill and providing a reference basis for the quality standard of the Qijudihuang pill. The method for detecting the fingerprint of the Qijudihuang pills, which is established for the first time, has good precision and repeatability, determines 15 common peaks, the RSD of the relative retention time of each common peak is less than or equal to 0.7 percent, the similarity evaluation results of multiple batches of Qijudihuang pills are all greater than 0.95, and the good stability of a test solution in 24 hours can be maintained.

The method provided by the invention can also carry out quantitative analysis on 9 chemical components (5-hydroxymethyl furfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B9) in the pill of Chinese wolfberry, chrysanthemum and rehmannia, so that one-time sample analysis is realized, and qualitative and quantitative analysis is completed at the same time.

The method provided by the invention can also confirm the attribution of chromatographic peaks of each single medicinal material in the pill of medlar, chrysanthemum and rehmannia, and the fingerprint embodies the characteristic peaks of medlar, prepared rehmannia root, tree peony bark, tuckahoe, chrysanthemum, wine dogwood fruit, yam and rhizoma alismatis. The method can realize effective monitoring from the source of raw materials, strictly control the quality of the raw materials, and ensure the relative stability of the effective component amount in the pill of Chinese wolfberry, chrysanthemum and rehmannia, thereby ensuring the safety and effectiveness of clinical medication. The established high performance liquid fingerprint spectrum method realizes the quality control of the whole formula of the Qijudihuang pill for the first time, does not identify single compounds or medicinal materials, has simple, stable and reliable method, good precision and repeatability, is convenient for quality control, and provides scientific test basis for better establishing an integral quality control evaluation system of the Qijudihuang pill. The fingerprint method of the Qijudihuang pill established by the invention is based on the pharmacodynamic substance basis of the medicinal materials, and realizes that one spectrum covers the pharmacodynamic substances of a plurality of medicinal materials by detecting in different wave bands.

Drawings

FIG. 1 shows the fingerprint of Qijudihuang pill of the present invention.

Fig. 2 shows the superimposed fingerprint of 10 batches of qiju dihuang pills in the invention.

FIG. 3 shows liquid chromatogram of QIJUDIHUANG pill sample, mixed reference, and medicinal materials, wherein A is QIJUDIHUANG pill sample, B is mixed reference, C is fructus Lycii, D is radix rehmanniae Preparata, E is cortex moutan, F is Poria, G is flos Chrysanthemi, H is Corni fructus, I is rhizoma Dioscoreae, and J is Alismatis rhizoma; the No. 2 peak is 5-hydroxymethylfurfural, the No. 3 peak is morroniside, the No. 4 peak is chlorogenic acid, the No. 5 peak is loganin, the No. 6 peak is paeoniflorin, the No. 7 peak is luteolin, the No. 9 peak is 3, 5-O-dicaffeoylquinic acid, the No. 13 peak is paeonol, and the No. 15 peak is 23-acetyl alisol B.

FIG. 4 shows liquid chromatogram of test solution and reference solution, wherein 1 is 5-hydroxymethylfurfural, 2 is morroniside, 3 is chlorogenic acid, 4 is loganin, 5 is paeoniflorin, 6 is luteolin, 7 is 3, 5-O-dicaffeoylquinic acid, 8 is paeonol, and 9 is 23-acetyl alisol B.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The reagents and equipment used in the following examples are as follows:

1. reagent

Methanol (analytical pure AR, chemical reagents ltd of the national drug group); acetonitrile (chromatographically pure, TEDIA corporation, usa); phosphoric acid (chromatographically pure, TEDIA corporation, usa); ultrapure water (produced by Milli-Q ultrapure water treatment system).

5-hydroxymethylfurfural (lot No. 111626-201912, mass fraction of 99.2%), morroniside (lot No. 111998-201703, mass fraction of 97.4%), chlorogenic acid (lot No. 110753-201817, mass fraction of 96.8%), loganin (lot No. 111640-201808, mass fraction of 99%), paeoniflorin (lot No. 110736-201943, mass fraction of 95.1%), luteolin (lot No. 111720-201810, mass fraction of 93.5%), 3, 5-O-dicaffeoylquinic acid (lot No. 111782-201807, mass fraction of 94.3%), paeonol (lot No. 110708-201908, mass fraction of 99.8%), 23-acetyl alisol B (lot No. 111846-201705, mass fraction of 99.7%), which are all purchased from the institute of food and drug assay.

Qijudihuang pills are provided by Shanghai and Huangyao limited company, and 10 batches of Qijudihuang pill powder are provided in the following Table 4.

TABLE 4 batch of Qiju Dihuang Wan

Numbering	Batch number	Numbering	Batch number
				S1	200323	S6	200601
S2	200324	S7	200603
				S3	200325	S8	200605
S4	200326	S9	200608
				S5	200327	S10	200610

The single medicinal material: fructus Lycii, radix rehmanniae Preparata, cortex moutan, Poria, flos Chrysanthemi, Corni fructus with wine, rhizoma Dioscoreae, and Alismatis rhizoma are all conventional Chinese medicinal materials, and can be purchased from market.

2. Instrument

Agilent 1260 high performance liquid chromatograph (OpenLAB CDS 2.1 chromatography workstation, G1312B binary pump, G1322A automatic degasser, G7116A column oven, G7115A DAD detector, G7129A autosampler, Agilent, usa); SB-5200DTD ultrasonic cleaning machine (Ningbo Xinzhi Biotech Co., Ltd.); analytical electronic balances of models AL204 and XS205 (Metler-Tolydo Meter Shanghai Co., Ltd.); Milli-Q Advantage A10 ultra pure water system (Mercury, Germany).

The process of the fingerprint spectrum detecting method of Qijudihuang pill provided by the invention is as follows:

1. sample pretreatment

Preparation of a test solution: pulverizing QIJUDIHUANG pill, precisely weighing 0.8g of QIJUDIHUANG pill powder, placing in 50mL conical flask with plug, precisely adding 24-26mL of 50-100% methanol water solution, weighing, sealing, ultrasonically extracting with supersonic instrument at power of 200-300W and frequency of 30-50kHz for 20-40min, cooling, weighing again, supplementing lost weight with 50-100% methanol water solution, collecting supernatant, and filtering with microporous membrane with pore diameter of 0.22 μm to obtain sample solution.

Preparation of control solutions: accurately weighing 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol, and 23-acetyl alisol B reference substances, and diluting in volumetric flask step by step. Firstly adding 50-100% methanol water solution to dissolve, and shaking up in volumetric flask with constant volume to obtain reference substance stock solution, and storing the reference substance stock solution in refrigerator at 4 deg.C in dark place. Adding 50-100% methanol water solution into the reference solution, diluting to constant volume, preparing into the desired reference solution, shaking the reference solution, filtering, and collecting filtrate. The content ranges of the components in the reference solution are as follows: 1.43-286.00 mu g/mL of 5-hydroxymethylfurfural, 1.80-359.21 mu g/mL of morroniside, 0.88-88.28 mu g/mL of chlorogenic acid, 2.16-431.21 mu g/mL of loganin, 2.15-430.61 mu g/mL of paeoniflorin, 1.38-138.38 mu g/mL of luteolin, 7.57-378.71 mu g/mL of 3, 5-O-dicaffeoylquinic acid, 9.76-976.40 mu g/mL of paeonol and 1.14-114.40 mu g/mL of 23-acetyl alisol B.

2. Chromatographic conditions

The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column; column temperature: 25-35 ℃; sample introduction amount: 1-20 μ L; the flow rate is 0.8-1.0 mL/min; the mobile phase is acetonitrile-0.05-0.2% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.2% phosphoric acid water solution; the analysis time is 85 min; gradient elution; the scanning wavelength is 200-350 nm.

As shown in table 1, the specific procedure of the gradient elution is:

3. Measurement of

And (3) respectively measuring the sample solution and the reference solution in the step (1) by adopting the high performance liquid chromatography under the chromatographic conditions in the step (2) to obtain the fingerprint of the sample solution and the fingerprint of the reference solution. And comparing the fingerprint of the test solution with the fingerprint of the reference solution, and identifying the corresponding characteristic peak in the fingerprint of the test solution according to the known characteristic peak in the fingerprint of the reference solution and the relative retention time, so as to perform attribution positioning on the index components in the fingerprint of the test solution and obtain the fingerprint of the Qiju-dihuang pill.

The quality detection method of the fingerprint spectrum of the Qijudihuang pill provided by the invention comprises the following steps:

the method comprises the steps of obtaining a fingerprint of the Qijudihuang pill by adopting the detection method of the fingerprint of the Qijudihuang pill, and comparing the similarity of the obtained fingerprint of the Qijudihuang pill with a standard fingerprint of the Qijudihuang pill obtained under the same fingerprint detection condition.

Specifically, when the measured fingerprint of the pill of Chinese wolfberry and rehmannia is compared with the standard fingerprint of the pill of Chinese wolfberry and rehmannia, the similarity is compared by adopting software 2009 of traditional Chinese medicine chromatography fingerprint similarity evaluation system issued by the committee of national pharmacopoeia.

The standard fingerprint of the pill of Chinese wolfberry and rehmannia comprises 15 common fingerprint peaks, wherein the 13 peak is taken as a positioning peak (S peak, retention time is 1.000), and the relative retention time of the other 14 common fingerprint peaks is sequentially No. 1 peak (0.133 +/-0.002), No. 2 peak (0.168 +/-0.002), No. 3 peak (0.354 +/-0.002), No. 4 peak (0.402 +/-0.003), No. 5 peak (0.470 +/-0.002), No. 6 peak (0.531 +/-0.002), No. 7 peak (0.693 +/-0.003), No. 8 peak (0.712 +/-0.004), No. 9 peak (0.776 +/-0.003), No. 10 peak (0.796 +/-0.003), No. 11 peak (0.829 +/-0.003), No. 12 peak (0.921 +/-0.002), No. 14 peak (1.206 +/-0.002) and No. 15 peak (1.002).

The standard fingerprint of the pill of Chinese wolfberry and rehmannia according to the preferred embodiment comprises 15 common fingerprint peaks, wherein the 13 peak is used as a positioning peak (S peak, retention time is 1.000), and the relative retention time of the other 14 common fingerprint peaks is sequentially 1 peak (0.133 +/-0.001), 2 peak (0.168 +/-0.001), 3 peak (0.354 +/-0.001), 4 peak (0.402 +/-0.002), 5 peak (0.470 +/-0.001), 6 peak (0.531 +/-0.001), 7 peak (0.693 +/-0.002), 8 peak (0.712 +/-0.003), 9 peak (0.206 +/-0.002), 10 peak (0.796 +/-0.002), 11 peak (0.829 +/-0.002), 12 peak (0.921 +/-0.001), 14 peak (1.206 +/-0.001) and 15 peak (1.001 +/-0.245).

As shown in fig. 4, the standard fingerprint of the pill of chinese wolfberry and rehmannia is compared with the fingerprint of a control solution, and the peak 1 is located and determined to be the fingerprint of 5-hydroxymethylfurfural, the peak 2 is the fingerprint of morroniside, the peak 3 is the fingerprint of chlorogenic acid, the peak 4 is the fingerprint of loganin, the peak 5 is the fingerprint of paeoniflorin, the peak 6 is the fingerprint of luteolin, the peak 7 is the fingerprint of 3, 5-O-dicaffeoylquinic acid, the peak 8 is the fingerprint of paeonol, and the peak 9 is the fingerprint of 23-acetylalisol B.

The process of the method for measuring the content of 9 components in the Qijudihuang pill provided by the invention is as follows:

1. preparation of a test solution: the preparation process of the test solution is the same as that of the test solution in the detection method of the fingerprint spectrum of the Qijudihuang pill.

2. Preparation of control solutions: the preparation process of the reference substance solution is the same as that of the detection method of fingerprint chromatogram of Qijudihuang pill.

3. And (3) determination: respectively measuring a test solution and a reference solution by adopting a high performance liquid chromatography with the same chromatographic conditions as in the detection method of the fingerprint of the Qijudihuang pill, and calculating the content of 9 components in the test solution by adopting an external standard method.

The external standard method is selected from one of an external standard one-point method or a standard curve method.

The process of the screening method of the fingerprint spectrums of the multiple medicinal materials in the Qijudihuang pill provided by the invention is as follows:

A) preparing a sample solution of a single medicinal material: preparing one or more of 8 medicinal material samples of fructus Lycii, radix rehmanniae Preparata, cortex moutan, Poria, flos Chrysanthemi, Corni fructus with wine, rhizoma Dioscoreae, and Alismatis rhizoma in QIJUDIHUANG pill according to preparation process of test solution of QIJUDIHUANG pill fingerprint chromatogram to obtain at least one single medicinal material sample solution;

B) and (3) determination: determining the sample solution of the single medicinal material by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions as the detection method of the fingerprint of the pill of Chinese wolfberry, chrysanthemum and rehmannia, and obtaining the fingerprint of the sample solution of the single medicinal material;

In the step A), every 200-300mL of the methanol aqueous solution is added with 1g of medlar, 1g of prepared rehmannia root, 1g of tree peony bark, 1g of tuckahoe, 1g of chrysanthemum, 1g of dogwood fruit wine, 1g of yam and 1g of rhizoma alismatis.

In the step D), in the single medicinal material sample solution, the fingerprint of the prepared rehmannia root sample solution comprises 1 common fingerprint peak, and the 1 common fingerprint peak is a No. 2 peak; the fingerprint of the moutan bark sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 6 peak and a No. 13 peak; the fingerprint spectrum of the chrysanthemum sample solution comprises 7 common fingerprint peaks, wherein the 7 common fingerprint peaks are a No. 4 peak, a No. 7 peak, a No. 8 peak, a No. 9 peak, a No. 10 peak, a No. 11 peak and a No. 12 peak; the fingerprint of the sample solution of the wine dogwood fruit comprises 4 common fingerprint peaks, wherein the 4 common fingerprint peaks are a No. 1 peak, a No. 2 peak, a No. 3 peak and a No. 5 peak; the fingerprint spectrum of the rhizoma alismatis sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 14 peak and a No. 15 peak; in the single medicinal material sample solution, the fingerprint spectrums of the medlar sample solution, the tuckahoe sample solution and the yam sample solution do not comprise common fingerprint peaks. The specific results are shown in Table 3. Among the above common fingerprint peaks, after comparison of the mixed reference containing 9 components in fig. 3, the peak 2 is determined to be 5-hydroxymethylfurfural, the peak 3 is morroniside, the peak 4 is chlorogenic acid, the peak 5 is loganin, the peak 6 is paeoniflorin, the peak 7 is luteolin, the peak 9 is 3, 5-O-dicaffeoylquinic acid, the peak 13 is paeonol, and the peak 15 is 23-acetylalisol B.

Example 1

1. Sample pretreatment

Preparation of a test solution: pulverizing QIJUDIHUANG pill, precisely weighing 0.8g powder of QIJUDIHUANG pill, placing into 50mL conical flask with plug, precisely adding 25mL of 80% methanol water solution, weighing, sealing, ultrasonically extracting with supersonic instrument at power of 250W and frequency of 40kHz for 30min, cooling, weighing again, supplementing loss weight with 80% methanol water solution, collecting supernatant, and filtering with microporous membrane with pore diameter of 0.22 μm to obtain test solution.

Preparation of control solutions: accurately weighing 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol, and 23-acetyl alisol B reference substances, and gradually diluting in a volumetric flask. Firstly adding 80% methanol water solution for dissolving, and uniformly shaking in a volumetric flask in a constant volume manner to obtain a reference substance storage solution, wherein the concentrations of all components in the reference substance storage solution are as follows: 0.286mg/mL of 5-hydroxymethylfurfural, 0.359mg/mL of morroniside, 0.177mg/mL of chlorogenic acid, 0.431mg/mL of loganin, 0.431mg/mL of paeoniflorin, 0.277mg/mL of luteolin, 0.379mg/mL of 3, 5-O-dicaffeoylquinic acid, 0.976mg/mL of paeonol and 0.229mg/mL of 23-acetyl alisol B. The reference stock solution was stored in a refrigerator at 4 ℃ in the dark. Adding 80% methanol water solution into the reference solution, diluting to constant volume, making into the desired reference solution, shaking the reference solution, filtering, and collecting the filtrate. The content ranges of the components in the reference solution are as follows: 1.43-286.00 mu g/mL of 5-hydroxymethylfurfural, 1.80-359.21 mu g/mL of morroniside, 0.88-88.28 mu g/mL of chlorogenic acid, 2.16-431.21 mu g/mL of loganin, 2.15-430.61 mu g/mL of paeoniflorin, 1.38-138.38 mu g/mL of luteolin, 7.57-378.71 mu g/mL of 3, 5-O-dicaffeoylquinic acid, 9.76-976.40 mu g/mL of paeonol and 1.14-114.40 mu g/mL of 23-acetyl alisol B.

The content range of each component in the reference solution can also be as follows: 28.60 mu g/mL of 5-hydroxymethylfurfural, 35.92 mu g/mL of morroniside, 17.66 mu g/mL of chlorogenic acid, 43.12 mu g/mL of loganin, 43.06 mu g/mL of paeoniflorin, 27.68 mu g/mL of luteolin, 37.87 mu g/mL of 3, 5-O-dicaffeoylquinic acid, 97.64 mu g/mL of paeonol and 22.88 mu g/mL of 23-acetyl alisol B.

2. Chromatographic conditions

The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is an Agilent 5TC-C18 chromatographic column (4.6mm multiplied by 250mm, 5 μm); column temperature: 30 ℃; sample injection amount: 10 mu L of the solution; the flow rate is 0.8-1.0 mL/min; the mobile phase is acetonitrile-0.1% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.1% phosphoric acid water solution; the analysis time is 85 min; gradient elution; the scanning wavelength is 210-334 nm.

As shown in table 1, the specific procedure of the gradient elution is:

70-85min, phase A: the volume ratio of the phase B is 95: 5-95: 5, the flow rate is 0.8mL/min

As shown in table 2, the specific procedure of the change of the scanning wavelength with the retention time is as follows:

scanning wavelength of 274nm for 0-17 min;

17-17.01min, the scanning wavelength is changed from 274nm to 240 nm;

17.01-34min, and the scanning wavelength is 240 nm;

34-34.01min, wherein the scanning wavelength is changed from 240nm to 334 nm;

34.01-68min, and the scanning wavelength is 334 nm;

68-68.01min, and changing the scanning wavelength from 334nm to 210 nm;

68.01-85min, and the scanning wavelength is 210 nm.

3. Measurement of

And (3) respectively measuring the sample solution and the reference solution in the step (1) by adopting the high performance liquid chromatography under the chromatographic conditions in the step (2) to obtain the fingerprint of the sample solution and the fingerprint of the reference solution. And the fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peak in the fingerprint of the test solution is identified through relative retention time according to the known characteristic peak in the fingerprint of the reference solution, so that the attribution positioning is carried out on the index components in the fingerprint of the test solution, and the fingerprint of the Qijudihuang pill is obtained.

Example 2

The fingerprint detection method of the Qijudihuang pills established in the embodiment 1 is adopted to detect 10 batches of Qijudihuang pills, so as to obtain the fingerprint of the test solution and the fingerprint of the reference solution, and the batch numbers of the 10 batches of Qijudihuang pills are shown in the table 4. The obtained fingerprint data of the test sample is imported into software 2009, published by the national pharmacopoeia committee, of the traditional Chinese medicine chromatography fingerprint similarity evaluation system, similarity comparison is carried out on the fingerprint data and standard fingerprints of the pill of Chinese wolfberry and rehmanniae under the same fingerprint detection condition, the similarity of the fingerprint of the pill of Chinese wolfberry and the standard fingerprint of the pill of Chinese wolfberry and rehmanniae is more than or equal to 0.95 as a standard, S1 samples in a table 4 are used as reference spectra, after multi-point correction, automatic matching is carried out, a reference spectrum is generated by an average method and is shown in a figure 1, and a superimposed spectrum is shown in a figure 2. The standard fingerprint spectrum of the totally calibrated pill of Chinese wolfberry, chrysanthemum and rehmannia comprises 15 shared fingerprint peaks which account for more than 90% of the total peak area, wherein the peak emergence time of the No. 13 peak is stable and moderate, so the No. 13 peak is selected as a positioning peak (S peak).

The retention time and the ratio of the retention peak area of each spectrum peak to the retention time and the retention peak area of the S peak in the same spectrum are calculated through the fingerprint obtained from 10 batches of Qijudihuang pills, and the obtained relative retention time and relative peak area are shown in Table 5. As can be seen from Table 5, the RSD of each common peak relative to the retention time is less than 0.7%, the RSD of the relative peak area is between 8 and 44%, and the similarity of 10 samples is above 0.95, which indicates that the components of 10 samples of Qiju Dihuang pill are relatively stable, indicating that the fingerprint is representative.

TABLE 5 relative retention time and relative peak area of common peaks

The specific standard fingerprint of the zhengqi tablet is shown in fig. 1, and as can be seen from fig. 1, the peak 13 is taken as a positioning peak (S peak, retention time is 1.000), and the relative retention times of the other 14 common fingerprint peaks are sequentially the peak 1 (0.133), the peak 2 (0.168), the peak 3 (0.354), the peak 4 (0.402), the peak 5 (0.470), the peak 6 (0.531), the peak 7 (0.693), the peak 8 (0.712), the peak 9 (0.776), the peak 10 (0.796), the peak 11 (0.829), the peak 12 (0.921), the peak 14 (1.206) and the peak 15 (1.245).

Comparing the standard fingerprint of the pill of Chinese wolfberry and rehmanniae radix with the fingerprint of a reference substance solution, identifying corresponding characteristic peaks in the standard fingerprint of the pill of Chinese wolfberry and rehmanniae radix by relative retention time according to known characteristic peaks in the fingerprint of the reference substance solution in fig. 4, and positioning and determining that the peak 1 is the fingerprint of 5-hydroxymethylfurfural, the peak 2 is the fingerprint of morroniside, the peak 3 is the fingerprint of chlorogenic acid, the peak 4 is the fingerprint of loganin, the peak 5 is the fingerprint of paeoniflorin, the peak 6 is the fingerprint of luteolin, the peak 7 is the fingerprint of 3, 5-O-dicaffeoylquinic acid, the peak 8 is the fingerprint of paeonol, and the peak 9 is the fingerprint of 23-acetyl alisol B.

Example 3

The method for detecting the fingerprint of the Qijudihuang pill is used for carrying out methodology verification, and the performance index result is as follows.

1. Precision degree

Taking 1 part of the Qijudihuang pill sample with the serial number of S2 (batch number: 200324), detecting according to the method for detecting the Qijudihuang pill fingerprint in the example 1, continuously sampling for 6 times, and inspecting the retention time of each main chromatographic peak in the fingerprint and the consistency of the peak area thereof, wherein the relative retention time RSD of 15 common peaks is less than or equal to 0.49 percent, and the relative peak area RSD is less than or equal to 2.38 percent. The results show that the instrument precision is good.

2. Repeatability of

Taking 6 parts of the sample of Qijudihuang pill with number S2 (batch number: 200324), detecting according to the method for detecting fingerprint of Qijudihuang pill in the above example 1, taking No. 13 peak (paeonol) as a reference peak, calculating the relative retention time of all the common peaks and the RSD of the relative peak area, and as a result, the RSD of 15 common peaks relative to the retention time is less than or equal to 0.2%, and the RSD of the relative peak area is less than or equal to 2.94%. The method has good repeatability and high accuracy.

3. Stability of

Taking 1 part of the sample of the pill of Chinese wolfberry and rehmanniae radix with the number of S2 (batch number: 200324), preparing a test solution according to the method for detecting the fingerprint of the pill of Chinese wolfberry and rehmanniae radix in the example 1, then respectively placing the test solution for 3h, 6h, 9h, 12h, 18h and 24h for detection, and calculating the relative retention time and the RSD of the relative peak area of each common peak by taking the No. 13 peak (paeonol) as a reference peak, wherein the RSD of the relative retention time of 15 common peaks is less than or equal to 0.29 percent, and the RSD% of the relative peak area is less than or equal to 2.94 percent. The sample in the invention is proved to have good detection stability within 24 h.

Example 4

By adopting the method for detecting the fingerprint of the Qijudihuang pills in the

embodiment

1, 10 batches of Qijudihuang pills are subjected to fingerprint analysis, the batch numbers of the 10 batches of Qijudihuang pills are shown in a table 4, and the detection result is shown in a table 2. Meanwhile, the overall similarity evaluation of 10 batches of samples of the Qijudihuang pill is carried out by adopting software 2009 of traditional Chinese medicine chromatogram fingerprint similarity evaluation system issued by the State pharmacopoeia Committee, the overall similarity can reflect the similarity of various components in different batches of samples in terms of types and relative contents, and the result is shown in Table 6. The results in Table 6 show that the similarity of 10 batches of Qijudihuang pills is more than or equal to 0.95, which indicates that the similarity of 10 batches of Qijudihuang pills is good, the difference between batches is small, and the fingerprint is suitable for the quality control of the Qijudihuang pills.

TABLE 610 batch of similarity evaluation results of Qijudihuang pills

	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	R
												S1	1.000
S2	0.998	1.000
												S3	0.989	0.989	1.000
S4	0.977	0.982	0.965	1.000
												S5	0.971	0.976	0.947	0.990	1.000
S6	0.973	0.979	0.954	0.995	0.998	1.000
												S7	0.995	0.994	0.990	0.976	0.962	0.967	1.000
S8	0.983	0.983	0.997	0.960	0.936	0.945	0.989	1.000
												S9	0.973	0.980	0.968	0.997	0.983	0.990	0.975	0.965	1.000
S10	0.995	0.994	0.993	0.974	0.961	0.966	0.998	0.991	0.975	1.000
												R	0.995	0.997	0.989	0.991	0.982	0.986	0.994	0.984	0.990	0.994	1.000

Example 5

1. Sample pretreatment

Preparation of a test solution: the procedure was the same as that for preparing the test solution in step 1 of example 1.

Preparation of control solutions: the same procedure was followed as in step 1 of example 1 to prepare a control solution.

2. Chromatographic conditions

The chromatographic conditions of the high performance liquid chromatography were the same as those of the high performance liquid chromatography in step 2 of example 1.

3. Measurement of

Preparing the prepared mixed reference substance solution into a series of mixed reference substance standard solutions with different concentrations by adopting an external standard method, carrying out sample injection analysis by adopting a high performance liquid chromatograph, and drawing a standard working curve. And then the obtained sample solution is subjected to sample injection analysis by a high performance liquid chromatograph, and the analysis result is substituted into the standard working curve, so that the content of 9 components in the sample solution is obtained.

Specifically, a series of reference substances with different volumes are respectively transferred to prepare a series of reference substance solutions with different concentrations, a high performance liquid chromatograph is adopted for sample injection analysis to obtain the linear relation between the content of 9 components in the reference substance solution and the peak area, each component chromatographic peak area corresponds to the corresponding content, a corresponding standard working curve is drawn, and the regression equation of each standard working curve is obtained through calculation. And detecting the sample solution by using a high performance liquid chromatograph, and substituting the chromatographic peak areas of the 9 components in the obtained sample solution into the regression equation of each standard working curve respectively to obtain the content of the corresponding component.

Example 6

Respectively preparing the 9 reference substances of different concentrations, precisely sucking 10 mu L, performing high performance liquid chromatography analysis by adopting the HPLC detection condition of the embodiment 5, drawing a standard curve by taking the concentration (mu g/mL) as a horizontal coordinate x and the peak area as a vertical coordinate y, and obtaining a regression equation, a correlation coefficient and a linear range, wherein the specific results are shown in a table 7.

As can be seen from Table 7, the regression equation has good linear relationship and correlation coefficient R when sample injection is performed in the corresponding concentration range²>0.9990. And repeatedly injecting a target substance response signal in the reference substance solution for 6 times by adopting a standard sample with the lowest concentration, carrying out HPLC analysis, and converting the S/N signal-to-noise ratio of more than or equal to 3 times as a detection Limit (LOD) and the S/N signal-to-noise ratio of more than or equal to 10 times as a quantification Limit (LOQ) into the content of the sample to obtain the target substance with the detection limit of 0.08-0.36 mu g/mL and the quantification limit of 0.15-0.72 mu g/mL, thereby having higher sensitivity.

TABLE 7 working curves and detection limits

Example 7

1. Precision degree

The same reference substance solution was sampled according to the chromatographic conditions of example 5, the sampling was repeated 6 times, the peak area data of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol, and 23-acetylalisol B was recorded, and the RSD of the peak areas of the above components was less than 1.35%, indicating good precision of the instrument.

2. Repeatability of

Taking a sample of Qijudihuang pill with the number of S2 (batch number: 200324), precisely weighing 6 parts, preparing the sample according to the preparation method of the test solution of the example 5, injecting samples according to the chromatographic conditions of the example 5, and recording peak area data of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B, wherein RSD of the peak areas of the components is less than 2.99%, which shows that the repeatability of the method is good.

3. Stability of

Taking a sample solution of a sample of Qijudihuang pills with the number of S2 (batch number: 200324), injecting samples for determination at 3h, 6h, 9h, 12h, 18h and 24h respectively according to the chromatographic conditions of the example 5, recording peak area data of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B, wherein the RSD of the peak areas of the components is less than 2.94%, which indicates that the stability of the sample solution in 24h is good.

4. Sample recovery rate

9 portions of the Qijudihuang pill sample (batch No. 200324) with the number S2 are precisely weighed, 3 portions of reference substance solutions (respectively corresponding to 80%, 100% and 120% of the original mass fraction) with low, medium and high mass concentrations are respectively added, 3 portions of each mass concentration are taken, the sample is prepared according to the preparation method of the test substance solution in the example 5, the sample is injected according to the chromatographic conditions in the example 5, and the sample adding recovery rate and the RSD of each component are calculated according to the measured amount and the added amount. The average recovery rates of the obtained 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B are 95.23-102.14%, and the RSD is 0.24-2.17%, which shows that the method has good accuracy.

Example 8

Taking 10 batches of Qijudihuang pill powder, preparing a sample according to the preparation method of the test solution in the example 5, injecting samples according to the chromatographic conditions in the example 5, recording peak areas, calculating the contents of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B by adopting an external standard method, and obtaining a result shown in a table 8, wherein the chromatogram of the sample and a reference substance is shown in a figure 4. The method can effectively measure the content of 9 components in the actual sample of the Qijudihuang pill, and has the advantages of simple operation, good applicability, and accurate and reliable result.

TABLE 810 measurement of the contents of Qijudihuang pills

Example 9

The sample solution was prepared by the sample pretreatment step described in example 1 above.

Taking 0.1g of medlar, prepared rehmannia root, tree peony bark, tuckahoe, chrysanthemum, wine dogwood fruit, yam and rhizoma alismatis respectively, and adopting the sample pretreatment step in the embodiment 1 to prepare sample solutions of 8 single medicinal materials. Wherein the addition amount of the methanol aqueous solution is 25 mL.

The fingerprint detection method of the pill of Chinese wolfberry and rehmannia according to the embodiment 1 is adopted to detect the sample solution and the 8 single medicinal material sample solutions respectively according to

items

2 and 3, so as to obtain the fingerprint of the sample solution and the 8 single medicinal material sample solutions respectively. The obtained test solution and the fingerprint of 8 single-medicinal-material sample solutions are introduced into software 2009, published by the national pharmacopoeia committee, of the traditional Chinese medicine chromatography fingerprint similarity evaluation system for analysis and processing, and meanwhile, the standard fingerprint of the Qiju-Dihuang pill is obtained by adopting the detection method of the fingerprint of the Qiju-Dihuang pill in the embodiment 1. The fingerprint spectrums of the sample solution to be tested and the 8 single medicinal material sample solutions are respectively compared with the standard fingerprint spectrum of the pill of Chinese wolfberry and rehmanniae, and the corresponding characteristic peaks of the 8 single medicinal material sample solutions in the standard fingerprint spectrum of the pill of Chinese wolfberry and rehmanniae are identified through relative retention time, so that the attribution positioning is carried out on the characteristic peaks in the fingerprint spectrums of the 8 single medicinal material sample solutions, and the specific result is shown in figure 3 and table 3.

As shown in fig. 3 and table 3, in the sample solution of the single herb, the fingerprint of the prepared rehmannia root sample solution includes 1 common fingerprint peak, and the 1 common fingerprint peak is peak No. 2; the fingerprint of the moutan bark sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 6 peak and a No. 13 peak; the fingerprint spectrum of the chrysanthemum sample solution comprises 7 common fingerprint peaks, wherein the 7 common fingerprint peaks are a No. 4 peak, a No. 7 peak, a No. 8 peak, a No. 9 peak, a No. 10 peak, a No. 11 peak and a No. 12 peak; the fingerprint of the sample solution of the wine dogwood fruit comprises 4 common fingerprint peaks, wherein the 4 common fingerprint peaks are a No. 1 peak, a No. 2 peak, a No. 3 peak and a No. 5 peak; the fingerprint spectrum of the alisma orientale sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 14 peak and a No. 15 peak; in the single medicinal material sample solution, the fingerprint spectrums of the medlar sample solution, the tuckahoe sample solution and the yam sample solution do not comprise common fingerprint peaks. In the common fingerprint peaks, after comparison of a mixed reference substance containing 9 components in fig. 3, the peak 2 is determined to be 5-hydroxymethylfurfural, the peak 3 is morroniside, the peak 4 is chlorogenic acid, the peak 5 is loganin, the peak 6 is paeoniflorin, the peak 7 is luteolin, the peak 9 is 3, 5-O-dicaffeoylquinic acid, the peak 13 is paeonol, and the peak 15 is 23-acetylalisol B.

The medlar, tuckahoe and yam do not comprise common peaks in 8 medicinal materials in the Qijudihuang pill, and the chemical characteristic peaks of other 5 medicinal materials are better reflected in the fingerprint spectrum and the attribution is confirmed.

In conclusion, the detection method and application of the fingerprint of the pill of Chinese wolfberry and rehmanniae, provided by the invention, establish an HPLC fingerprint and a multi-component simultaneous quantitative analysis method of the pill of Chinese wolfberry and rehmanniae, determine 15 common characteristic peaks, analyze common peak sources, identify 9 components, and quantitatively analyze the 9 chemical components (5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, luteolin, 3, 5-O-dicaffeoylquinic acid, paeonol and 23-acetyl alisol B). Similarity analysis is adopted to evaluate the fingerprint spectrums of different batches of the Qijudihuang pills, and the content is analyzed, so that the method is stable and reliable, has good precision and repeatability, and provides scientific experimental basis for better establishing an integral quality control evaluation system of the Qijudihuang pills. . Therefore, the present invention overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A method for detecting a fingerprint spectrum of Qijudihuang pills comprises the following steps:

1) preparing a test solution: adding methanol water solution into the pill sample of Chinese wolfberry, chrysanthemum and rehmannia, carrying out ultrasonic extraction, and filtering supernatant to obtain a test solution;

3) and (3) determination: respectively measuring a test solution and a reference solution by adopting a high performance liquid chromatography with the same chromatographic condition to obtain a fingerprint of the test solution and a fingerprint of the reference solution, comparing the fingerprint of the test solution with the fingerprint of the reference solution, and performing attribution positioning on index components in the fingerprint of the test solution, thereby obtaining a fingerprint of the Qiju-Dihuang pill;

in the step 3), in the high performance liquid chromatography, a chromatographic column is a C18 chromatographic column; the flow rate is 0.8-1.0 mL/min; the mobile phase is acetonitrile-0.05-0.2% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.2% phosphoric acid water solution; the analysis time is 85 min; gradient elution;

the specific procedure of the gradient elution is as follows:

70-85min, phase A: the volume ratio of the phase B is 95: 5-95: 5, the flow rate is 0.8 mL/min;

the specific procedure for scanning wavelength variation with retention time is:

scanning wavelength of 274nm for 0-17 min;

17-17.01min, the scanning wavelength is changed from 274nm to 240 nm;

17.01-34min, and the scanning wavelength is 240 nm;

34-34.01min, wherein the scanning wavelength is changed from 240nm to 334 nm;

34.01-68min, and the scanning wavelength is 334 nm;

68-68.01min, and changing the scanning wavelength from 334nm to 210 nm;

68.01-85min, and the scanning wavelength is 210 nm.

2. The method for detecting the fingerprint of Qijudihuang pills according to claim 1, wherein the step 1) comprises any one or more of the following conditions:

A1) the ratio of the weight of the added sample of the Qijudihuang pill to the volume of the added methanol water solution is 4: 120-130 g/mL;

A2) the ultrasonic extraction time is 20-40 min;

A3) the power of the ultrasonic extraction is 200-300W, and the frequency of the ultrasonic extraction is 30-50 kHz;

A4) the methanol water solution is methanol water solution with volume percentage concentration of more than or equal to 50 percent.

3. The use of the fingerprint spectrum detection method of Qijudihuang pills according to any one of claims 1-2 in quality detection of components in the Qijudihuang pills.

4. A quality detection method of Qijudihuang pill comprises obtaining fingerprint of Qijudihuang pill by the detection method of fingerprint of Qijudihuang pill as claimed in any one of claims 1-2, and comparing the obtained fingerprint with standard fingerprint of Qijudihuang pill obtained under the same fingerprint detection condition.

5. The method for detecting the quality of Qijudihuang pills according to claim 4, wherein the standard fingerprint of the Qijudihuang pills is obtained by the same conditions as the method for detecting the fingerprint of the Qijudihuang pills according to any one of claims 1 to 2, the standard fingerprint of the Qijudihuang pills comprises 15 common fingerprint peaks, the peak 13 is used as the positioning peak S peak, the retention time is 1.000, and the relative retention times of the other 14 common fingerprint peaks are 0.131 to 0.135 of the peak 1, 0.166 to 0.170 of the peak 2, 0.352 to 0.356 of the peak 3, 0.399 to 0.405 of the peak 4, 0.468 to 0.472 of the peak 5, 0.529 to 0.533 of the peak 6, 0.690 to 0.696 of the peak 7, 0.38708 to 0.716 of the peak 8, 0.773 to 0.779, 0.793 to 0.799 of the peak 10, 0.793 to 0.799 of the peak 7, 0.832 to 0.826, 0.919 of the peak 0.12 to 0.204 to 25.204 of the peak 1 to 25, Peak 15 from 1.243 to 1.247.

6. A method for screening fingerprints of multiple medicinal materials in Qijudihuang pills comprises the following steps:

A) preparing a sample solution of a single medicinal material: preparing any one or more of 8 medicinal material samples of medlar, prepared rehmannia root, tree peony bark, tuckahoe, chrysanthemum, wine dogwood fruit, yam and rhizoma alismatis in the pill of medlar, chrysanthemum and rehmannia root according to the step 1) of the detection method of fingerprint of pill of medlar, chrysanthemum and rehmannia root of any one of claims 1 to 2, and respectively obtaining at least one single medicinal material sample solution;

B) and (3) determination: measuring the sample solution of the single medicinal material by adopting the high performance liquid chromatography with the same chromatographic conditions in the step 3) of the fingerprint detection method of the Qijudihuang pill according to any one of claims 1-2 to obtain the fingerprint of the sample solution of the single medicinal material;

C) obtaining a standard fingerprint: obtaining a standard fingerprint spectrum of the Qijudihuang pill by adopting the same steps as the detection method of the fingerprint spectrum of the Qijudihuang pill in any one of claims 1-2;

7. The method for screening fingerprints of multiple herbs in Qijudihuang pill as claimed in claim 6, wherein the amount of fructus Lycii, radix rehmanniae Preparata, cortex moutan, Poria, flos Chrysanthemi, Corni fructus, rhizoma Dioscoreae and Alismatis rhizoma is 1g, respectively, per 200-300mL of methanol aqueous solution, respectively.