CN112285264A

CN112285264A - Detection method of fingerprint spectrum of Zhibai Dihuang pill and application thereof

Info

Publication number: CN112285264A
Application number: CN202011391810.6A
Authority: CN
Inventors: 田禾苗; 温方方; 姜鹏; 詹常森; 张正光
Original assignee: Shanghai Hutchison Pharmaceuticals Co ltd
Current assignee: Shanghai Hutchison Pharmaceuticals Co ltd
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2021-01-29

Abstract

The invention provides a method for detecting fingerprint spectra of Zhibai Dihuang pills. The invention also provides application of the detection method of the fingerprint of the Zhibai Dihuang pill in quality detection of components in the Zhibai Dihuang pill and a quality detection method thereof. The invention further provides a method for measuring the content of 5 components in the Zhibai Dihuang pill. The invention further provides a method for screening the fingerprint spectrums of the multiple medicinal materials in the Zhibai Dihuang pill. The invention provides a detection method of fingerprint of Zhibai Dihuang pills and application thereof, which establishes a high-performance liquid phase fingerprint of the Zhibai Dihuang pills, can carry out quantitative analysis on 5 chemical components in the Zhibai Dihuang pills, and can carry out attribution confirmation on chromatographic peaks of each single medicinal material in the Zhibai Dihuang pills, can effectively monitor from a raw material source, strictly control the quality of the raw medicinal material, and ensure that the amount of the effective components in the Zhibai Dihuang pills is relatively stable, thereby ensuring the safety and effectiveness of clinical medication.

Description

Detection method of fingerprint spectrum of Zhibai Dihuang pill 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 Zhibai Dihuang pills and application thereof, and particularly relates to a detection method of a fingerprint of Zhibai Dihuang pills and application thereof in quality detection.

Background

Zhibai Dihuang Wan is a recorded variety in 2020 edition Chinese pharmacopoeia. Is brown black water-honeyed pill prepared from 8 Chinese medicinal materials including anemarrhena rhizome, phellodendron bark, prepared rehmannia root, Chinese yam, cornus officinalis, poria cocos, tree peony bark and alisma orientale through steps of sieving, mixing, adding refined honey, adding water, pilling and drying. Has the effects of nourishing yin and lowering fire, and is mainly used for treating yin deficiency and fire excess, tidal fever and night sweat, dry mouth and pharyngalgia, tinnitus and spermatorrhea, scanty and brownish urine and other symptoms clinically.

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 Zhibai Dihuang pills is lacked. Therefore, it is necessary to establish a fingerprint spectrum and a multi-component content measurement method of the Zhibai Dihuang pill, which can complete qualitative and quantitative analysis of the components in the Zhibai Dihuang pill so as to effectively control the quality of the Zhibai Dihuang pill.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a detection method of an Zhibai Dihuang pill fingerprint spectrum and application thereof, which adopts pretreatment of optimized conditions and a high performance liquid chromatography method to establish the high performance liquid chromatography fingerprint spectrum of the Zhibai Dihuang pill, and highlights contributions of different types of chemical components to a Zhibai Dihuang pill fingerprint spectrum system from different side surfaces, thereby realizing detection of all chemical components of the Zhibai Dihuang pill, more comprehensively reflecting the current situation of each component in the Zhibai Dihuang pill, and providing a reference basis for overall control and evaluation of the quality of the Zhibai Dihuang pill.

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

1) preparation of a test solution: dissolving the sample of the pill of Anemarrhena, phellodendron and rehmannia with an organic solvent, carrying out ultrasonic extraction, cooling, filtering, and taking a subsequent filtrate to obtain a test solution;

2) preparation of control solutions: adding organic solvent into the morroniside, loganin, mangiferin, berberine hydrochloride and paeonol reference substance, and dissolving with ultrasound to obtain 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 Zhibai Dihuang pill.

Preferably, in the step 1), the rhizoma anemarrhenae, cortex phellodendri and radix rehmanniae pill sample is a crude drug powder sample of the rhizoma anemarrhenae, cortex phellodendri and radix rehmanniae pill obtained by pulverizing and sieving crude drugs of the rhizoma anemarrhenae, cortex phellodendri and radix rehmanniae pill.

More preferably, the screen is a size four screen.

Preferably, in step 1), the sample of Zhibai Dihuang pill is prepared before the sample of Zhibai Dihuang pill is prepared into the test solution⁶⁰Co-gamma ray irradiation.

More preferably, the⁶⁰The dosage of Co-gamma ray irradiation is 3-10 KGy. Further preferably, the⁶⁰The dose of Co-gamma irradiation was 6 KGy.

More preferably, the⁶⁰The maximum absorbed dose of Co-gamma ray irradiation is less than or equal to 6 KGy.

The above-mentioned⁶⁰Co-gamma ray radiation sterilization technology means utilization⁶⁰The gamma ray generated by Co can kill the microbes on the surface and inside of the material. The Chinese medicine is taken from root and rhizome and grows in the strainIn a soil environment with abundant and complicated microorganisms, the medicinal materials still carry a certain amount of microorganisms after being cleaned and cut. Especially, the Chinese patent medicine which is directly powdered for use has strict requirements on the initial bacteria content of the medicinal materials, otherwise the bacteria content of the finished product exceeds the standard. The irradiation with a certain radiation amount can effectively reduce the content of microorganisms in the traditional Chinese medicine, and simultaneously reduce the microbial load, thereby achieving the effect of sterilizing the traditional Chinese medicine. Therefore, it is necessary to perform irradiation sterilization on crude drug powder of raw medicinal materials of a Chinese medicinal preparation, thereby further reducing the microbial load.

Preferably, in step 1), the ratio of the weight (g) of the added sample of rhizoma anemarrhenae, phellodendri and rehmannia glutinosa to the volume (mL) of the added organic solvent is 1: 15-25.

More preferably, the ratio of the weight (g) of the sample of Zhibai Dihuang pills added to the volume (mL) of the organic solvent added is 1: 20.

preferably, in step 1), the organic solvent is 50-100% methanol. The 50-100% methanol is 50-100% methanol water solution.

More preferably, the organic solvent is 80% methanol.

Preferably, in the step 1), the sample of the Zhibai Dihuang pill needs to be precisely weighed after the organic solvent is added.

Preferably, in the step 1), the ultrasonic extraction time is 15-60 min. More preferably, the ultrasound extraction time is 30 min.

Preferably, in step 1), the power of the ultrasonic extraction is 80-120W, and the frequency of the ultrasonic extraction is 30-50 kHz. More preferably, the power of the ultrasonic extraction is 100W, and the frequency of the ultrasonic extraction is 40 kHz.

Preferably, in step 1), the weight loss is compensated with an organic solvent after the cooling.

Preferably, in the step 1), the filtration mode is a filter membrane filtration mode. More preferably, the pore size of the filter is 0.45 μm.

Preferably, in step 1), the subsequent filtrate is obtained after the filtrate gives up the primary filtrate.

Preferably, in the step 2), the CAS number of the morroniside is 25406-64-8, the CAS number of the loganin is 18524-94-2, the CAS number of the mangiferin is 4773-96-0, the CAS number of the berberine hydrochloride is 633-65-8, and the CAS number of the paeonol is 552-41-0.

Preferably, in step 2), the content ranges of the components in the control solution are as follows: the content of morroniside is 0.3990mg/mL, the content of loganin is 0.3530mg/mL, the content of mangiferin is 0.2562mg/mL, the content of berberine hydrochloride is 0.2946mg/mL, and the content of paeonol is 0.7552 mg/mL.

Preferably, in step 2), the organic solvent is 50-100% methanol. The 50-100% methanol is 50-100% methanol water solution.

More preferably, the organic solvent is 80% methanol.

Preferably, in the step 2), the ultrasonic extraction time is 5-15min in the ultrasonic dissolution.

Preferably, in the step 2), in the ultrasonic dissolution, the power of the ultrasonic extraction is 50-100W, and the frequency of the ultrasonic extraction is 10-40 kHz.

Preferably, in step 2), the control solution is stored in a refrigerator at 4 ℃ in the dark.

Preferably, in the step 3), when the control solution is measured, a certain volume of the control solution is diluted after the organic solvent is added.

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), and the packing material is octadecylsilane chemically bonded silica.

Preferably, in step 3), the detector in the high performance liquid chromatography is a photodiode array detector (DAD).

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 0.8-1.2 mL/min. More preferably, in the high performance liquid chromatography, the flow rate is 1.0 mL/min.

Preferably, in the step 3), the detection wavelength in the high performance liquid chromatography is 200-400 nm. More preferably, in the high performance liquid chromatography, the detection wavelength is 254 nm.

Preferably, in the step 3), the mobile phase in the high performance liquid chromatography 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 100 min; gradient elution.

More preferably, in the high performance liquid chromatography, the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, wherein, the phase A is acetonitrile, and the phase B is 0.1% phosphoric acid aqueous solution; the analysis time is 100 min; gradient elution.

The 0.05-0.2% phosphoric acid aqueous solution is 0.05-0.2% phosphoric acid aqueous solution by volume percentage. The 0.1% phosphoric acid aqueous solution is 0.1% phosphoric acid aqueous solution in volume percentage.

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

0-5min, phase A: the volume ratio of the phase B is 5: 95-7: 93;

5-20min, phase A: the volume ratio of the phase B is 7: 93-11: 89;

20-35min, phase A: the volume ratio of the phase B is 11: 89-11: 89;

35-60min, phase A: the volume ratio of the phase B is 11: 89-25: 75;

60-80min, phase A: the volume ratio of the phase B is 25: 75-50: 50;

80-95min, phase A: the volume ratio of the phase B is 50: 50-80: 20;

95-100min, phase A: the volume ratio of the phase B is 80: 20-95: 5.

TABLE 1 gradient elution procedure

The second aspect of the invention provides an application of a detection method of fingerprint spectra of Zhibai Dihuang pills in quality detection of components in the Zhibai Dihuang pills.

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

Preferably, when the measured fingerprint of the Zhibai Dihuang pill is compared with the standard fingerprint of the Zhibai Dihuang pill, software of 2012 version of the evaluation system for similarity of traditional Chinese medicine chromatogram fingerprint issued by the State pharmacopoeia Committee is adopted for comparison. More preferably, the similarity between the fingerprint of the Zhibai Dihuang pill measured by the invention and the standard fingerprint of the Zhibai Dihuang pill is more than or equal to 0.991.

More preferably, when the fingerprint common peak of the Zhibai Dihuang pill fingerprint is matched with the standard fingerprint of the Zhibai Dihuang pill, the generation method of the comparison map adopts a median method, and the time window width is 0.1.

Preferably, the standard fingerprint of the Zhibai Dihuang pill is obtained by adopting the same conditions as the detection method of the Zhibai Dihuang pill fingerprint, and comprises 12 common fingerprint peaks, wherein the 11 peak is taken as a reference peak (S peak, retention time is 1.0000 +/-0.0010), and the relative retention time of the other 11 common fingerprint peaks is 1 peak (0.0472 +/-0.0010), 2 peak (0.0622 +/-0.0010), 3 peak (0.2971 +/-0.0015), 4 peak (0.3349 +/-0.0020), 5 peak (0.4790 +/-0.0030), 6 peak (0.5245 +/-0.0035), 7 peak (0.6263 +/-0.0030), 8 peak (0.7971 +/-0.0025), 9 peak (0.8467 +/-0.0025), 10 peak (0.9924 +/-0.0010) and 12 peak (1.0986 +/-0.0030).

More preferably, the standard fingerprint of Zhibai Dihuang Wan comprises 12 common fingerprint peaks, wherein 11 common fingerprint peaks are used as reference peaks (S peak, retention time is 1.0000 + -0.0001), and the relative retention time of other 11 common fingerprint peaks is sequentially 1 peak (0.0472 + -0.0001), 2 peak (0.0622 + -0.0002), 3 peak (0.2971 + -0.0009), 4 peak (0.3349 + -0.0013), 5 peak (0.4790 + -0.0023), 6 peak (0.5245 + -0.0028), 7 peak (0.6263 + -0.0024), 8 peak (0.7971 + -0.0020), 9 peak (0.8467 + -0.0019), 10 peak (0.9924 + -0.0002) and 12 peak (1.0986 + -0.0023).

The specific data of the standard fingerprint of the pill of Anemarrhena, Phellodendron and rehmannia is shown in figure 1.

More preferably, the standard fingerprint of the Zhibai Dihuang pill is compared with the fingerprint of a reference solution, and the No. 3 peak is located and determined to be the fingerprint of morroniside, the No. 5 peak is the fingerprint of loganin, the No. 6 peak is the fingerprint of mangiferin, the No. 11 peak is the fingerprint of berberine hydrochloride, and the No. 12 peak is the fingerprint of paeonol.

The fourth aspect of the invention provides a method for measuring the content of 5 components in Zhibai Dihuang pills, which comprises the following steps:

a) preparation of a test solution: the method is the same as the step 1) of the detection method of the fingerprint of the Zhibai Dihuang pill;

b) preparation of control solutions: the detection method is the same as the step 2) of the fingerprint spectrum detection method of the Zhibai Dihuang 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 in the fingerprint detection method of the Zhibai Dihuang pill, and calculating the content of 5 components in the test solution by adopting an external standard method.

Preferably, the external standard method is as follows: respectively transferring a series of reference substance solutions with different volumes in the step b) to prepare a series of solutions with different concentrations, carrying out sample injection analysis by adopting a high performance liquid chromatograph to obtain the linear relation between the content of 5 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 the 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 5 components in the obtained sample solution into the regression equation of each standard working curve respectively to obtain the content of the corresponding component.

More preferably, in the standard working curve, the peak area is taken as the ordinate, and the content of each control solution is taken as the abscissa.

The fifth aspect of the invention provides a method for screening fingerprint spectrums of a plurality of medicinal materials in Zhibai Dihuang 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 Corni, radix rehmanniae Preparata, rhizoma anemarrhenae, cortex Phellodendri, cortex moutan, rhizoma Dioscoreae, Poria, and rhizoma Alismatis in ZHIBAIDIHUANG pill according to step 1) of fingerprint detection method of ZHIBAIDIHUANG 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 Zhibai Dihuang 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 Zhibai Dihuang pill by adopting the same steps as the detection method of the fingerprint spectrum of the Zhibai Dihuang pill;

D) and (3) quality detection: comparing the fingerprint of the single medicinal material sample solution with the standard fingerprint of Zhibai Dihuang pill, and identifying the corresponding characteristic peak of the single medicinal material sample solution in the standard fingerprint of Zhibai Dihuang 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 dogwood is dried ripe pulp of Cornus officinalis sieb. The Radix Rehmanniae Preparata is rhizome of rehmannia glutinosa or rehmannia glutinosa Radix Praeparata of Scrophulariaceae. The rhizoma anemarrhenae is dried rhizome of Anemarrhena asphodeloides Bunge of Liliaceae. The cortex Phellodendri is dried bark of Phellodendron chinense Schneid of Rutaceae. The Cortex Moutan Moutan Cortex is dried root bark of Paeonia suffruticosa Andr. The rhizoma Dioscoreae is dried rhizome of Dioscorea opposita Thunb of Dioscoreaceae. The Poria is dried fungus of Poria cocos (Schw.) wolf. The Alismatis rhizoma is dried tuber of Alisma orientale (Sam.) J uzep or Alisma plantata gaaquatica Linn.

Preferably, in the step D), the attribution of the characteristic peaks of the measured fingerprint spectrum of the single-drug sample solution and the standard fingerprint spectrum of the Zhibai Dihuang pill is positioned, the analysis and the processing are performed by adopting software of 2012 edition of "traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation System" issued by the State pharmacopoeia Commission, and the attribution of the characteristic peaks of the respective single-drug of the Zhibai Dihuang pill is confirmed by the relative retention time of the respective characteristic peaks on the standard fingerprint spectrum of the Zhibai Dihuang pill.

Preferably, in the step D), the fingerprint of the sample solution for preparing dogwood comprises 5 common fingerprint peaks, and the 5 common fingerprint peaks are peak 1, peak 2, peak 3, peak 5 and peak 8.

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.

Preferably, in the step D), in the sample solution of the single herb, the fingerprint of the sample solution of rhizoma anemarrhenae includes 2 common fingerprint peaks, and the 2 common fingerprint peaks are peak 4 and peak 6.

Preferably, in the step D), in the sample solution of the single medicinal material, the fingerprint of the sample solution of phellodendron amurense includes 3 common fingerprint peaks, and the 3 common fingerprint peaks are a peak 7, a peak 10 and a peak 11.

Preferably, in the step D), the fingerprint of the moutan bark sample solution in the single herb sample solution includes 3 common fingerprint peaks, and the 3 common fingerprint peaks are peak No. 8, peak No. 9 and peak No. 12.

Preferably, in the step D), the fingerprint spectrums of the yam sample solution, the poria cocos sample solution and the rhizoma alismatis sample solution do not include a common fingerprint peak.

Among the common fingerprint peaks, the peak 3 is determined to be the fingerprint peak of morroniside, the peak 5 is determined to be the fingerprint peak of loganin, the peak 6 is determined to be the fingerprint peak of mangiferin, the peak 11 is determined to be the fingerprint peak of berberine hydrochloride, and the peak 12 is determined to be the fingerprint peak of paeonol.

The water used in the invention is pure water.

As mentioned above, the invention provides a detection method of fingerprint of Zhibai Dihuang Wan and application thereof, which adopts pretreatment of optimized reaction conditions and a high performance liquid chromatography method to establish the high performance liquid chromatography fingerprint of Zhibai Dihuang Wan, and highlights the contribution of different chemical components to the fingerprint system of Zhibai Dihuang Wan from different side faces, thereby realizing the detection of the full formula chemical components of the Zhibai Dihuang Wan and providing reference basis for the quality standard of the Zhibai Dihuang Wan. The detection method of the fingerprint of the Zhibai Dihuang pill established for the first time has good precision and repeatability, determines 12 common peaks, has similarity evaluation results of a plurality of batches of Zhibai Dihuang pills of more than 0.991, and can keep good stability of a test solution within 24 hours.

The method provided by the invention can also carry out quantitative analysis on 5 chemical components (morroniside, loganin, mangiferin, berberine hydrochloride and paeonol) in the Zhibai Dihuang pill, realize one-time sample analysis and simultaneously complete qualitative and quantitative analysis.

The method provided by the invention can also be used for treating Zhibai Dihuang pills⁶⁰Co-gamma ray irradiation effectively reduces the content of microorganisms in raw medicinal powder of Zhibai Dihuang pills, thereby ensuring the medication safety of the Zhibai Dihuang pills.

The method provided by the invention can also confirm the attribution of a plurality of single medicinal material chromatographic peaks in the Zhibai Dihuang pill, and the fingerprint embodies the characteristic peaks of prepared dogwood, prepared rehmannia root, rhizoma anemarrhenae, phellodendron, moutan bark, Chinese yam, tuckahoe 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 Zhibai Dihuang pill, thereby ensuring the safety and effectiveness of clinical medication. The high performance liquid fingerprint method established by the invention realizes the quality control of the whole formula of the Zhibai Dihuang 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 Zhibai Dihuang pill.

Drawings

Fig. 1 shows the fingerprint of Zhibai Dihuang Wan in the invention, wherein, 3: morroniside; 5: loganin; 6: mangiferin; 11: berberine hydrochloride (S); 12: paeonol.

FIG. 2 shows the spectrum peak assignment chart of Zhibai Dihuang pill of the present invention.

FIG. 3 shows the fingerprint of the sample of Zhibai Dihuang Wan in the present invention.

Fig. 4 shows chromatograms 4A and 4B of the comparison product of Zhibai Dihuang pill and the sample of Zhibai Dihuang pill of 200301 batch in the invention, wherein fig. 4A is the chromatogram of the comparison product of Zhibai Dihuang pill, 1: morroniside; 2: loganin; 3: mangiferin; 4: berberine hydrochloride; 5: paeonol; FIG. 4B is a chromatogram of a 200301 batch sample of Zhibai Dihuang pill,

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

Morroniside (batch number: 111998-.

Methanol, phosphoric acid (chromatographically pure, TEDIA corporation, usa); ultrapure water (produced by Milli-Q ultrapure water treatment system).

Pill of rhizoma anemarrhenae, cortex Phellodendri and rehmanniae radix provided by Shanghai and Huang pharmaceutical Co Ltd in 4 batches (with batch numbers of 200301, 200302, 200303 and 200304)

The single medicinal material: the processed dogwood, the prepared rehmannia root, the rhizoma anemarrhenae, the phellodendron, the moutan bark, the Chinese yam, the poria cocos and the rhizoma alismatis are all conventional Chinese medicinal materials provided by Shanghai and Huang medicine Limited companies and can be purchased from the market.

2. Instrument for measuring the position of a moving object

Agilent 1260 high performance liquid chromatograph (Agilent OpenLAB CDS ChemStation workstation, Quaternary Pump System model G1311C, Standard Autosampler model G1329B, column oven model G1316A, diode array Detector model G4212B, Agilent Corp.); one electron day of 204 ten thousandths of AL and one electron balance of ten thousandths of X205BDU (METTLER-TOLEDO meter overseas ltd); DFY-500 type high-speed Chinese medicinal pulverizer (Dade Chinese medicine machinery, Inc. of Wenling Ridge, Zhejiang); TDL-40B bench centrifuge (Shanghai' an pavilion scientific instruments factory); DHG-9123A type electric hot blast drying oven (shanghai-heng science instruments ltd); SB-5200 ultrasonic cleaning machine (Ningbo Xinzhi Biotech Co., Ltd.); Mill-Q Advantage A10 ultrapure water preparation system (Michlobo Millipore Shanghai trade Co., Ltd.).

The process of the fingerprint spectrum detecting method of Zhibai Dihuang Wan provided by the invention is as follows:

1. sample pretreatment

Preparation of a test solution: pulverizing rhizoma anemarrhenae, cortex Phellodendri, radix rehmanniae Preparata and sieving with a sieve of four numbers to obtain rhizoma anemarrhenae, radix rehmanniae Preparata and radix rehmanniae Preparata powder,at a dose of 3-10KGy⁶⁰The irradiation of Co-gamma ray is carried out,⁰the maximum absorbed dose of Co-gamma ray irradiation is less than or equal to 6 KGy. Taking 1.0g of crude drug powder of Bo-Dihuang pill, placing in a conical flask with a plug, adding 15-25ml of 50-100% methanol water solution precisely, weighing, sealing the plug, ultrasonic extracting with an ultrasonic instrument at power of 80-120W and frequency of 30-50kHz for 15-60min, cooling, weighing again, supplementing loss weight with 50-100% methanol water solution, taking supernatant, filtering with microporous membrane with pore diameter of 0.45 μm, and taking the filtrate to obtain sample solution.

Preparation of control solutions: precisely weighing morroniside, loganin, mangiferin, berberine hydrochloride and paeonol reference substances, placing in a volumetric flask, adding 50-100% methanol water solution, ultrasonic dissolving at power of 50-100W and frequency of 10-40kHz, shaking to constant volume in the volumetric flask to obtain reference substance solution, and storing in a refrigerator at 4 deg.C in dark place. The content ranges of the components in the reference solution are as follows: the content of morroniside is 0.3990mg/mL, the content of loganin is 0.3530mg/mL, the content of mangiferin is 0.2562mg/mL, the content of berberine hydrochloride is 0.2946mg/mL, and the content of paeonol is 0.7552 mg/mL. When the reference substance solution is used for measurement, a certain volume of the reference substance solution is diluted by adding 50-100% methanol water solution.

2. Chromatographic conditions

The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column; the detector is a photodiode array detector (DAD); column temperature: 25-35 ℃; sample introduction amount: 1-20 μ L; the flow rate is 0.8-1.2 mL/min; the detection wavelength is 200-400 nm; 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 100 min; gradient elution.

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

0-5min, phase A: the volume ratio of the phase B is 5: 95-7: 93;

5-20min, phase A: the volume ratio of the phase B is 7: 93-11: 89;

20-35min, phase A: the volume ratio of the phase B is 11: 89-11: 89;

35-60min, phase A: the volume ratio of the phase B is 11: 89-25: 75;

60-80min, phase A: the volume ratio of the phase B is 25: 75-50: 50;

80-95min, phase A: the volume ratio of the phase B is 50: 50-80: 20;

95-100min, phase A: the volume ratio of the phase B is 80: 20-95: 5.

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 of the chromatographic conditions in the step (2) to obtain the fingerprint of the sample solution and the fingerprint of the reference solution. 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 of the index component in the fingerprint of the test solution is performed, and the fingerprint of the Zhibai Dihuang pill is obtained.

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

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

Specifically, when the measured fingerprint of Zhibai Dihuang pill is compared with the standard fingerprint of Zhibai Dihuang pill, software of 2012 version of evaluation system for similarity of traditional Chinese medicine chromatogram fingerprint issued by the State Committee of pharmacopoeia is adopted for comparison.

The standard fingerprint of Zhibai Dihuang Wan comprises 12 common fingerprint peaks, wherein 11 peaks are used as reference peaks (S peak, retention time is 1.0000 +/-0.0010), and the relative retention time of other 11 common fingerprint peaks is sequentially 1 peak (0.0472 +/-0.0010), 2 peak (0.0622 +/-0.0010), 3 peak (0.2971 +/-0.0015), 4 peak (0.3349 +/-0.0020), 5 peak (0.4790 +/-0.0030), 6 peak (0.5245 +/-0.0035), 7 peak (0.6263 +/-0.0030), 8 peak (0.7971 +/-0.0025), 9 peak (0.8467 +/-0.0025), 10 peak (0.9924 +/-0.0010) and 12 peak (1.0986 +/-0.0030).

As shown in fig. 1, the standard fingerprint of the pill of hinoki and rehmannia is compared with the fingerprint of a reference solution, and the peak 3 is determined to be the fingerprint of morroniside, the peak 5 is the fingerprint of loganin, the peak 6 is the fingerprint of mangiferin, the peak 11 is the fingerprint of berberine hydrochloride, and the peak 12 is the fingerprint of paeonol.

The process of the method for measuring the content of 5 components in the Zhibai Dihuang 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 fingerprint detection method of Zhibai Dihuang Wan.

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

3. And (3) determination: respectively measuring the test solution and the reference solution by high performance liquid chromatography with the same chromatographic conditions as those in the fingerprint detection method of the Zhibai Dihuang pill, and calculating the content of 5 components in the test solution by an external standard method.

The process of the screening method of the fingerprint spectrums of the multiple medicinal materials in the Zhibai Dihuang 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 Corni, radix rehmanniae Preparata, rhizoma anemarrhenae, cortex Phellodendri, cortex moutan, rhizoma Dioscoreae, Poria, and rhizoma Alismatis in ZHIBAIDIHUANG pill according to fingerprint sample solution preparation process 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 fingerprint detection method of the Zhibai Dihuang pill to obtain the fingerprint of the sample solution of the single medicinal material;

In the step D), in the sample solution of the single medicinal material, as shown in figure 2, the fingerprint spectrum of the dogwood sample solution comprises 5 common fingerprint peaks, wherein the 5 common fingerprint peaks are a peak 1, a peak 2, a peak 3, a peak 5 and a peak 8; the fingerprint of the prepared rehmannia root sample solution comprises 1 common fingerprint peak, wherein the 1 common fingerprint peak is a No. 2 peak; the fingerprint spectrum of the rhizoma anemarrhenae sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 4 peak and a No. 6 peak; the fingerprint spectrum of the phellodendron amurense sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 7 peak, a No. 10 peak and a No. 11 peak; the fingerprint of the moutan bark sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are No. 8 peak, No. 9 peak and No. 12 peak. The fingerprint spectrums of the yam sample solution, the tuckahoe sample solution and the rhizoma alismatis sample solution do not comprise common fingerprint peaks.

Among the common fingerprint peaks, after comparison by a reference substance containing 5 components, the peak 3 is determined to be the fingerprint peak of morroniside, the peak 5 is the fingerprint peak of loganin, the peak 6 is the fingerprint peak of mangiferin, the peak 11 is the fingerprint peak of berberine hydrochloride, and the peak 12 is the fingerprint peak of paeonol.

Example 1

1. Sample pretreatment

Preparation of a test solution: pulverizing the 200301 crude drugs of ZHIBAIDIHUANG pill, sieving with a fourth sieve to obtain crude drug powder, and processing at a dose of 6KGy⁶⁰The irradiation of Co-gamma ray is carried out,⁰the maximum absorbed dose of Co-gamma irradiation was 6 KGy. Taking 1.0g of crude drug powder of Bo-Dihuang pill, placing in a conical flask with a plug, precisely adding 20ml of 80% methanol aqueous solution, weighing, sealing the plug, and using an ultrasonic instrument at a power of 100W,Ultrasonic extracting at 40kHz for 30min, cooling, weighing again, adding 80% methanol water solution to reduce weight, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution.

Preparation of control solutions: precisely weighing morroniside, loganin, mangiferin, berberine hydrochloride and paeonol reference substances, placing in a volumetric flask, adding 80% methanol water solution, ultrasonic dissolving at power of 75W and frequency of 25kHz, shaking to constant volume in the volumetric flask to obtain reference substance solution, and storing in a refrigerator at 4 deg.C in dark place. The content ranges of the components in the reference solution are as follows: the content of morroniside is 0.3990mg/mL, the content of loganin is 0.3530mg/mL, the content of mangiferin is 0.2562mg/mL, the content of berberine hydrochloride is 0.2946mg/mL, and the content of paeonol is 0.7552 mg/mL. When the control solution is measured, a series of solutions with different volumes of the control solution diluted by 80% methanol water solution can be taken.

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 mu m), and the filler is octadecylsilane chemically bonded silica; the detector is a photodiode array detector (DAD); column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; the flow rate is 1.0 mL/min; the detection wavelength is 254 nm; 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 100 min; gradient elution; .

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

0-5min, phase A: the volume ratio of the phase B is 5: 95-7: 93;

5-20min, phase A: the volume ratio of the phase B is 7: 93-11: 89;

20-35min, phase A: the volume ratio of the phase B is 11: 89-11: 89;

35-60min, phase A: the volume ratio of the phase B is 11: 89-25: 75;

60-80min, phase A: the volume ratio of the phase B is 25: 75-50: 50;

80-95min, phase A: the volume ratio of the phase B is 50: 50-80: 20;

95-100min, phase A: the volume ratio of the phase B is 80: 20-95: 5.

3. measurement of

Example 2

The method for detecting fingerprints of Zhibai Dihuang pills established in the embodiment 1 is adopted to detect a plurality of batches of Zhibai Dihuang pills, so as to obtain the fingerprints of a test solution and the fingerprints of a reference solution, the obtained fingerprint data of the test solution is introduced into software of 2012 edition of traditional Chinese medicine chromatography fingerprint similarity evaluation system issued by the State pharmacopoeia Committee, similarity comparison is carried out on the fingerprints of the Zhibai Dihuang pills obtained under the same fingerprint detection condition, the similarity between the fingerprints of the Zhibai Dihuang pills and the standard fingerprints of the Zhibai Dihuang pills is more than or equal to 0.991, a median method is adopted as a reference generation method, the time window width is 0.2, and a superimposed spectrum is shown in FIG. 3. The standard fingerprint spectrum of the co-calibrated Zhibai Dihuang pill comprises 12 common fingerprint peaks, wherein the 11 number is a reference peak (S peak).

The specific standard fingerprint of Zhibai Dihuang Wan is shown in FIG. 1, and it can be seen from FIG. 1 that the 11 peak is used as the reference peak (S peak, retention time is 1.0000 + -0.0001), and the relative retention times of the other 11 common fingerprint peaks are sequentially the 1 peak (0.0472 + -0.0001), the 2 peak (0.0622 + -0.0002), the 3 peak (0.2971 + -0.0009), the 4 peak (0.3349 + -0.0013), the 5 peak (0.4790 + -0.0023), the 6 peak (0.5245 + -0.0028), the 7 peak (0.6263 + -0.0024), the 8 peak (0.7971 + -0.0020), the 9 peak (0.8467 + -0.0019), the 10 peak (0.9924 + -0.0002), and the 12 peak (1.0986 + -0.0023).

Comparing the standard fingerprint of the Zhibai Dihuang pill with that of a reference substance solution, identifying the corresponding characteristic peak in the standard fingerprint of the Zhibai Dihuang pill according to the known characteristic peak in the fingerprint of the reference substance solution in figure 1 by relative retention time, and positioning and determining that the No. 3 peak is the fingerprint of morroniside, the No. 5 peak is the fingerprint of loganin, the No. 6 peak is the fingerprint of mangiferin, the No. 11 peak is the fingerprint of berberine hydrochloride, and the No. 12 peak is the fingerprint of paeonol.

Example 3

The detection method of the fingerprint of the Zhibai Dihuang pill is verified by methodology, and the performance index result is as follows.

1. Precision degree

Taking 1 part of 200302 batches of Zhibai Dihuang pill samples, detecting according to the detection method of the Zhibai Dihuang pill fingerprint in the embodiment 1, continuously sampling for 6 times, recording a chromatogram, and inspecting the relative retention time of a specified common peak in the fingerprint, wherein the RSD is between 0.12 and 0.34 percent, and the relative retention area RSD is between 0.18 and 0.33 percent, and the result shows that the method has good precision.

2. Repeatability of

Taking 6 samples of the Zhibai Dihuang pills of 200302 batches, detecting according to the detection method of the Zhibai Dihuang pills fingerprint in the embodiment 1, taking the No. 11 peak (berberine hydrochloride) as a reference peak, calculating the relative retention time of all the common peaks and the RSD of the relative peak area, and determining that the RSD of the relative retention time of the common peaks specified by the fingerprint is between 0.18 and 0.29 percent and the RSD of the relative retention peak area is between 1.32 and 2.26 percent, which shows that the method has good repeatability and higher accuracy.

3. Stability of

Taking 1 part of 200302 batches of Zhibai Dihuang pill samples, respectively placing for 0h, 3h, 6h, 9h, 12h and 24h for detection after preparing a test sample solution according to the detection method of the Zhibai Dihuang pill fingerprint chromatogram in the embodiment 1, taking a No. 11 peak (berberine hydrochloride) as a reference peak, calculating to obtain the relative retention time of each common peak and the RSD of the relative peak area, wherein the RSD of the relative retention time of the common peak specified by the fingerprint chromatogram is 0.19-0.28%, and the RSD of the relative retention peak area is 1.69-2.35%, which indicates that the samples have good detection stability in 24 h.

Example 4

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

As shown in fig. 4, a series of reference solutions with different volumes are respectively transferred by an external standard method to prepare a series of solutions with different concentrations, and a standard working curve is drawn by sampling analysis of a high performance liquid chromatograph. 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 5 components in the sample solution is obtained.

Specifically, a series of reference substance solutions with different volumes are respectively transferred to prepare a series of solutions with different concentrations, a high performance liquid chromatograph is adopted for sample injection analysis to obtain the linear relation between the content of 5 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 5 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 5

Respectively preparing the 5 reference substances of different concentrations, precisely sucking 10 mu L, performing high performance liquid chromatograph analysis by adopting the HPLC detection conditions of the embodiment 4, 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 Table 2.

As can be seen from Table 2, the regression equation has good linear relationship when sample injection is carried out in the corresponding concentration range, and the correlation coefficient r²>0.9995. The concentration of the reference substance is taken as a limit of quantitation (LOQ) when the signal-to-noise ratio of the peak area is 10 times (S/N is 10), and the concentration of the reference substance is taken as a limit of quantitation (LOD) when the signal-to-noise ratio of the peak area is 3 times (S/N is 3), and the concentration is converted into the content of the sample, so that the target substance has high sensitivity.

TABLE 2 working curves and detection limits

Example 6

1. Precision degree

Sample injection is carried out on 1 part of 200302 batches of Zhibai Dihuang pills according to the chromatographic conditions of the example 4, sample injection is repeated for 6 times, chromatogram is recorded, and the RSD (n is 6) of the morroniside, the loganin, the mangiferin, the berberine hydrochloride and the paeonol are respectively 1.30 percent, 1.07 percent, 0.88 percent, 0.40 percent and 0.37 percent according to peak areas, which indicates that the precision of the instrument is good.

2. Repeatability of

A200302 batch sample of Zhibai Dihuang Wan is precisely weighed as 6 parts, the sample is prepared according to the preparation method of the test solution of the example 4, the sample injection is carried out according to the chromatographic conditions of the example 4, and the chromatogram is recorded. The average content of 5 components of morroniside, loganin, mangiferin, berberine hydrochloride and paeonol is 1.1654, 0.8549, 0.7918, 0.8397 and 2.2199mg/g respectively; RSD were 1.10%, 3.57%, 1.37%, and 0.77%, respectively (n ═ 6), and the reproducibility was good.

3. Stability of

Sample solutions of 200302 batches of Zhibai Dihuang pills are taken, sample introduction and measurement are carried out for 0h, 3h, 6h, 9h, 12h and 24h according to the chromatographic conditions of the example 4, chromatograms are recorded, peak areas are measured, and the results show that the RSD (n ═ 6) of the morroniside, loganin, mangiferin, berberine hydrochloride and paeonol are 1.19%, 1.04%, 0.74%, 0.48% and 0.30% respectively, which indicates that the sample solutions are stable within 24 h.

4. Sample recovery rate

9 parts of samples of Zhibai Dihuang pills of 200302 batches are precisely weighed to be 0.50g, newly prepared reference substance solutions (containing 0.3028mg/ml of morroniside, 0.2230mg/ml of loganin, 0.2480mg/ml of mangiferin, 0.2014mg/ml of berberine hydrochloride and 0.3684mg/ml of paeonol) with different volumes are precisely added according to 80%, 100% and 120% of inherent contents of the morroniside, loganin, mangiferin, berberine hydrochloride and paeonol in crude drug powder respectively, 3 parts of each mass concentration are taken, samples are prepared according to the preparation method of the test solution of the embodiment 4, samples are injected according to the chromatographic conditions of the embodiment 4, and the chromatogram is recorded. The recovery rates of the 5 effective components at different addition ratios were calculated. Results of the morroniside, loganin, mangiferin, berberine hydrochloride and paeonol sample recovery rates ranged from 95.29-104.49%, the average recovery rates were 99.0%, 99.2%, 99.0%, 99.6% and 98.9%, and the RSD (n ═ 3) was 1.38%, 2.28%, 1.72%, 3.83% and 2.40%, respectively, and the results are shown in table 3. Indicating that the accuracy of the method is good.

TABLE 3 sample Loading recovery test results (n ═ 3)

Example 7

4 batches of raw medicinal powder of Zhibai Dihuang pills (batch numbers: 200301, 200302, 200303, 200304) are taken, irradiated by 6kGy, samples are prepared according to the preparation method of the test sample solution of the example 4, sample injection is carried out according to the chromatographic conditions of the example 4, the peak area is recorded, and the contents of the morroniside, loganin, mangiferin, berberine hydrochloride and paeonol are calculated by adopting an external standard method, and the results are shown in Table 4. The method can effectively determine the content of 5 components in the sample of Zhibai Dihuang pill, and has the advantages of simple operation, good applicability, and accurate and reliable result.

TABLE 44 determination of content of Zhibai Dihuang Wan batches

Example 8

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

Respectively taking the dogwood, the prepared rehmannia root, the rhizoma anemarrhenae, the phellodendron, the moutan bark, the Chinese yam, the tuckahoe and the rhizoma alismatis, and adopting the sample pretreatment step in the embodiment 1 to prepare 8 sample solutions of single medicinal materials.

By adopting the

steps

2 and 3 of the method for detecting the fingerprint of the Zhibai Dihuang Wan in the embodiment 1, the sample solution to be tested and the sample solution of 8 single medicinal materials are respectively measured, and the fingerprint of the sample solution to be tested and the fingerprint of the sample solution of 8 single medicinal materials are respectively obtained. The obtained test solution and the fingerprints of the 8 single-drug sample solutions are introduced into software of 2012 edition "traditional Chinese medicine chromatography fingerprint similarity evaluation system" issued by the national pharmacopoeia committee for analysis and processing, and meanwhile, the standard fingerprint of the Zhibai Dihuang pill is obtained by adopting the detection method of the fingerprint of the Zhibai Dihuang pill in the embodiment 1. Comparing the fingerprint spectrums of the sample solution to be tested and the 8 single medicinal material sample solutions with the standard fingerprint spectrum of the Zhibai Dihuang pill respectively, and identifying the corresponding characteristic peaks of the 8 single medicinal material sample solutions in the standard fingerprint spectrum of the Zhibai Dihuang pill through relative retention time, thereby performing attribution positioning on the characteristic peaks in the fingerprint spectrums of the 8 single medicinal material sample solutions, wherein the specific result is shown in figure 2.

As can be seen from fig. 2, in the sample solution of the single medicinal material, the fingerprint of the sample solution of the dogwood comprises 5 common fingerprint peaks, wherein the 5 common fingerprint peaks are a peak number 1, a peak number 2, a peak number 3, a peak number 5 and a peak number 8; the fingerprint of the prepared rehmannia root sample solution comprises 1 common fingerprint peak, wherein the 1 common fingerprint peak is a No. 2 peak; the fingerprint spectrum of the rhizoma anemarrhenae sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 4 peak and a No. 6 peak; the fingerprint spectrum of the phellodendron amurense sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 7 peak, a No. 10 peak and a No. 11 peak; the fingerprint of the moutan bark sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are No. 8 peak, No. 9 peak and No. 12 peak. The fingerprint spectrums of the yam sample solution, the tuckahoe sample solution and the rhizoma alismatis sample solution do not comprise common fingerprint peaks. Among the common fingerprint peaks, after comparison by a reference substance containing 5 components, the peak 3 is determined to be the fingerprint peak of morroniside, the peak 5 is the fingerprint peak of loganin, the peak 6 is the fingerprint peak of mangiferin, the peak 11 is the fingerprint peak of berberine hydrochloride, and the peak 12 is the fingerprint peak of paeonol.

Therefore, the chemical characteristic peaks of the 8 medicinal materials in the Zhibai Dihuang pill are better reflected in the fingerprint spectrum and are confirmed to belong to the same herb.

In conclusion, the detection method and the application of the fingerprint of the Zhibai Dihuang pill provided by the invention establish an HPLC fingerprint and multi-component simultaneous quantitative analysis method of the Zhibai Dihuang pill, determine 12 common characteristic peaks, analyze common peak sources, identify 5 components, and quantitatively analyze the 5 chemical components (morroniside, loganin, mangiferin, berberine hydrochloride and paeonol). 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 Zhibai Dihuang 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. Any person skilled in the art can modify or change the above-mentioned 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 detection method of fingerprint spectra of Zhibai Dihuang Wan comprises the following steps:

3) and (3) determination: respectively measuring the test solution and the 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 the fingerprint of the Zhibai Dihuang pill.

2. The method for detecting fingerprint of Zhibai Dihuang Wan as claimed in claim 1, wherein in step 1) or 2), any one or more of the following conditions are included:

A1) in the step 1), the sample of Zhibai Dihuang pill is prepared before the sample solution of the test article is prepared⁶⁰Co-gamma ray irradiation of said⁶⁰The dose of Co-gamma ray irradiation is 3-10 KGy;

A2) in the step 1), the ratio of the weight of the sample of the Zhibai Dihuang pill to the volume of the organic solvent is 1: 15-25 g/mL;

A3) in the step 1), the ultrasonic extraction time is 15-60 min;

A4) in the step 1), the power of ultrasonic extraction is 80-120W, and the frequency of ultrasonic extraction is 30-50 kHz;

A5) in the step 2), in the ultrasonic dissolution, the ultrasonic extraction time is 5-15 min;

A6) in the step 2), in the ultrasonic dissolution, the power of ultrasonic extraction is 50-100W, and the frequency of ultrasonic extraction is 10-40 kHz;

A7) in the step 1) or 2), the organic solvent is 50-100% methanol.

3. The method for detecting fingerprint of Zhibai Dihuang Wan as claimed in claim 1, wherein in step 3), the chromatographic conditions of the HPLC are as follows: the chromatographic column is a C18 chromatographic column; the detector is a photodiode array detector; the column temperature is 25-35 ℃; the sample amount is 1-20 mu L; the flow rate is 0.8-1.2 mL/min; the detection wavelength is 200-400 nm; 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 100 min; gradient elution.

4. The method for detecting fingerprint of Zhibai Dihuang Wan as claimed in claim 3, wherein the specific procedure of gradient elution is as follows: 0-5min, phase A: the volume ratio of the phase B is 5: 95-7: 93; 5-20min, phase A: the volume ratio of the phase B is 7: 93-11: 89; 20-35min, phase A: the volume ratio of the phase B is 11: 89-11: 89; 35-60min, phase A: the volume ratio of the phase B is 11: 89-25: 75; 60-80min, phase A: the volume ratio of the phase B is 25: 75-50: 50; 80-95min, phase A: the volume ratio of the phase B is 50: 50-80: 20; 95-100min, phase A: the volume ratio of the phase B is 80: 20-95: 5.

5. the use of the fingerprint detection method of Zhibai Dihuang Wan as claimed in any one of claims 1-4 in quality detection of components in Zhibai Dihuang Wan.

6. A quality detection method of ZHIBAIDIHUANG pill comprises obtaining ZHIBAIDIHUANG pill fingerprint by the method of any one of claims 1-4, and comparing the obtained ZHIBAIDIHUANG pill fingerprint with standard fingerprint of ZHIBAIDIHUANG pill obtained under the same fingerprint detection condition.

7. The method for detecting the quality of Zhibai Dihuang Wan according to claim 6, wherein the standard fingerprint of Zhibai Dihuang Wan is obtained under the same conditions as the method for detecting the fingerprint of Zhibai Dihuang Wan according to any one of claims 1 to 4, wherein the standard fingerprint of Zhibai Dihuang Wan comprises 12 common fingerprint peaks, wherein the 11 peak is taken as the S peak of the reference peak, the retention time is 0.9990 to 1.0010, and the relative retention time of the other 11 common fingerprint peaks is sequentially 1 peak 0.0462 to 0.0482, 2 peak 0.0612 to 0.0632, 3 peak 0.2956 to 0.2986, 4 peak 0.3329 to 0.3369, 5 peak 0.4760 to 0.4820, 6 peak 0.5210 to 0.5280, 7 peak 0.6233 to 0.6293, 8 peak 0.7946 to 0.7996, 9 peak 48 to 0.8492, 10 peak 390.9914 to 0.9934, and 12 peak 1.0956 to 1.1016.

8. A method for measuring the content of 5 components in Zhibai Dihuang Wan comprises the following steps:

a) preparation of a test solution: the same as the step 1) of the method for detecting fingerprint of Zhibai Dihuang Wan as claimed in any one of claims 1 to 4;

b) preparation of control solutions: the same as the step 2) of the method for detecting fingerprint of Zhibai Dihuang Wan as claimed in any one of claims 1-4;

c) and (3) determination: respectively measuring the test solution in the step a) and the reference solution in the step b) by using a high performance liquid chromatography with the same chromatographic conditions as in the fingerprint detection method of Zhibai Dihuang pill in any one of claims 1 to 4, and calculating the content of 5 components in the test solution by using an external standard method.

9. A method for screening fingerprint spectrums of multiple medicinal materials in Zhibai Dihuang 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 dogwood, prepared rehmannia root, common anemarrhena rhizome, amur corktree bark, tree peony bark, common yam rhizome, Indian buead and oriental waterplantain rhizome in the Zhibai Dihuang pill according to the step 1) of the detection method of the fingerprint of the Zhibai Dihuang pill in any one of claims 1 to 4 to respectively obtain at least one single medicinal material sample solution;

B) and (3) determination: determining the sample solution of the single medicinal material by using the high performance liquid chromatography with the same chromatographic conditions as in the step 3) of the fingerprint detection method of Zhibai Dihuang Wan as claimed in any one of claims 1 to 4, thereby obtaining the fingerprint of the sample solution of the single medicinal material;

C) obtaining a standard fingerprint: obtaining a standard fingerprint of the Zhibai Dihuang pill by the same steps as the detection method of the fingerprint of the Zhibai Dihuang pill as claimed in any one of claims 1 to 4;

10. The method for screening fingerprints of multiple medicinal materials in Zhibai Dihuang Wan as claimed in claim 9, wherein step D) includes any one or more of the following conditions:

B1) the fingerprint of the dogwood sample solution comprises 5 common fingerprint peaks, wherein the 5 common fingerprint peaks are a No. 1 peak, a No. 2 peak, a No. 3 peak, a No. 5 peak and a No. 8 peak;

B2) the fingerprint of the prepared rehmannia root sample solution comprises 1 common fingerprint peak, wherein the 1 common fingerprint peak is a No. 2 peak;

B3) the fingerprint spectrum of the rhizoma anemarrhenae sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 4 peak and a No. 6 peak;

B4) the fingerprint spectrum of the phellodendron amurense sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 7 peak, a No. 10 peak and a No. 11 peak;

B5) the fingerprint spectrum of the moutan bark sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 8 peak, a No. 9 peak and a No. 12 peak;

B6) the fingerprint spectrums of the yam sample solution, the tuckahoe sample solution and the rhizoma alismatis sample solution do not comprise common fingerprint peaks.